Prepared by
Mazen M. El Zaharna
Prof. Fadel A. Sharief
First Edition 2008
A shy person does learn, nor should a
short-tempered person teach
Contents
Laboratory Safety
Exercise 1
1
3
Mitotic Cell Division
Exercise 2
7
Agarose Gel Electrophoresis
Exercise 3
13
Human Metaphase Chromosomes
Exercise 4
17
Human Chromosome Identification by G-Banding (karyotyping)
Exercise 5
24
Extraction of Human DNA
Exercise 6
28
Polymerase Chain Reaction (PCR)
Exercixe 7
32
Restriction Enzyme Digestion & Southern Blotting of DNA
Exercixe 8
38
Plasmid DNA Isolation
Exercixe 9
41
Transformation of Escherichia coli
Exercixe 10
SDS-PAGE
45
Laboratory Safety
Safety in the Laboratory Should always be in your mind. Throughout this
manual Safety recommendations are given, below are some general
consideration that anyone in a laboratory should know.
•
General laboratory safety precaution.
1. Follow all instructions carefully. Use special care when you see the
word CAUTION in your laboratory instructions. Follow the safety
instructions given by your instructor.
2. Determine the location of Fire Extinguishers, Chemical safety showers
and Eye washers, Chemical Spill Kits, and alternative exit routes for lab
evacuation.
3. Remember that smoking, eating, or drinking in the lab room is totally
prohibited.
4. Lab coats should be always worn during the work in the lab.
5. Wear safety goggles when using dangerous chemicals, hot liquids, or
burners.
6. Any chemicals spilled on the hands or other parts of the skin should be
washed off immediately with a plenty of running water.
7. If you have an open skin wound, be sure that it is covered with a water
proof bandage.
8. Never work alone in the laboratory.
9. Keep your work area clean & dry.
10. Turn of all electrical equipment, water, and gas when it is not in use,
especially at the end of the laboratory period.
11. Report all chemicals spills or fluids to your instructor immediately for
proper clean up.
•
Special precautions for working with heat or fire:
1. Never leave a lighted Bunsen burner unattended. When an object is
removed from the heat & left to cool, it should be placed where it is
shielded from contact.
2. Inflammable liquid bottles should not be left open, not dispensed near
a naked flame, hot electric element or electric motor.
3. Use test tube holders to handle hot laboratory equipments.
4. When you are heating something in a container such as a test tube,
always point the open end of the container away from yourself &
others.
•
Special precautions for working with chemicals
1. Never taste or touch substances in the laboratory without specific
instructions.
2. Never smell substances in the laboratory without specific instructions.
3. Use materials only from containers that are properly labeled.
4. Wash your hand after working with chemicals.
5. Do not add water to acid. Instead, dilute the acid by adding it to water.
Practical Genetics 1st ed. 2008
1
•
Special precautions for working with electrical equipment.
1. Make sure the area under & around the electrical equipment is dry.
2. Never touch electrical equipment with wet hands.
3. Make sure the area surrounding the electrical equipment is free of
flammable materials.
4. Turn off all power switches before plugging an appliance into an outlet.
•
Special Precaution for working with Glasswares and other
laboratory equipments.
1. Become familiar with the names and appearance of all the laboratory
equipments you will use.
2. Never use broken or chipped glassware.
3. Make sure that all glasswares are clean before you using it.
4. Do not pick up broken glass with your bare hands. Use a pan and a
brush.
5. If a Mercury thermometer breaks, do not touch the mercury. Notify
your instructor immediately.
6. Use care handling all sharp equipments, such as scalpels and
dissecting needles.
•
Special precautions for working with live or preserved specimens.
1. If live animals are used treat them gently. Follow instructions for their
proper care.
2. Always wash your hands after working with live or preserved
organisms.
3. Do not open Petri dishes containing live cultures unless you are
directed to do so.
4. Detergents (detol 5 – 10%) should be used to sterilize and clean
benches, glassware and equipment.
1. Safety cabinet should be used while working with microbes.
2. Disposable items should be collected and autoclaved.
Practical Genetics 1st ed. 2008
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1
Exercise 1: Mitotic Cell Division
Objectives:
• Learn a staining procedure to identify the stages of mitosis in onion root tip.
• To differentiate between the different stages of mitosis.
• Calculate the mitotic index.
Introduction:
The growth and development of every organism depends on the precise
replication of the genetic material during each cell division. Cell division is the
process by which cells reproduce themselves, involves both division of the
cell’s nucleus (karyokinesis) and division of the cytoplasm (cytokinesis).
There are two types of nuclear division: mitosis and meiosis. New body
(somatic) cells are formed by mitosis. Each cell division produces two new
daughter cells with the same number and kind of chromosomes as the parent
cell. The formation of male and female gametes in animal cells or spores in
plant cells is by meiosis. Gametes and spores will have half the chromosome
number of the parent cells.
Stages of mitosis
Interphase
Interphase, which begins when cell division
ends and continues until the beginning of the
next round of division, is organized into three
phases. G1, S and G2 (Figure 1.1). The
chromatin in this phase is undifferentiated in
the heavily-stained nucleus. Before the cell
enters the mitosis phase, it first undergoes a
synthesis or S phase where each
chromosome is duplicated and consists of two
sister chromatids joined together by a
centromere. Centromeres are crucial to
segregation of the daughter chromatids during
mitosis. Now, the nucleus and cell increase in
size, and chromosomes are fully extended.
The cell is preparing for the beginning of
mitosis.
Figure 1.1. Stages of the cell cycle.
Mitosis
Mitosis is the next phase of the cell cycle. It is essentially the same whether
considering a simple plant or a highly evolved organism, such as a human
being. The major function of mitosis is to accurately and precisely replicate
genetic information, or chromosomes, so each daughter cell contains the
same information. The process of mitosis is an ongoing event that can be
segmented into several identifiable stages. In order, these stages are:
prophase, metaphase, anaphase, and telophase. Cytokinesis (Figure 1.2),
the actual process of cell division, occurs during telophase. In plants such as
Practical Genetics 1st ed. 2008
Exercise 1
Mitotic Cell Division
the onion, this is seen as the formation of the cell plate between the two
daughter cells.
Prophase
In prophase, dramatic changes begin to occur within the
nucleus of the cell. Chromosomes become thicker, shorter,
and easily visible when stained under the light microscope.
Two “sister chromatids” join near their middle at a
structure called the centromere. The nucleolus and the
nuclear membrane disappear. The mitotic apparatus the
spindle, begins to organize within the cell. Microtubules
are slender rods of protein responsible for pulling replicated
chromosomes towards each half of the cell.
Metaphase
During this period, chromosomes become aligned at
midpoint or equator between poles of the cell and are at
their thickest and shortest structure. They are easily
identified as two longitudinally double sister chromatids.
Chromatids are connected (at their centromeres) to the
spindle apparatus, which has formed between the two
centrioles located at the poles of the cell. In many plants,
the centrioles are absent. The spindle is still present,
however, and the plant chromosomes are similarly attached
to the spindle microtubular fibers.
Anaphase
In this short phase, sister chromatids begin to separate and
migrate to the poles. Once the two chromatids separate,
each is called a chromosome. For humans, with a diploid
number of 46 chromosomes, there will be 46 chromosomes
moving toward each pole. Onions have 16 diploid
chromosomes and, therefore 16 chromosomes move to
each pole. During anaphase there is a quantitative, equal
segregation of the diploid number of chromosomes into two
developing nuclei at the poles of the anaphase cell.
Prophase
Metaphase
Early anaphase
Late anaphase
Telophase
Telophase and Cytokinesis
Figure 1.2. The phases of
The final mitotic phase of the cell cycle is recognized by the
mitotic division.
formation of two new nuclei encompassing the daughter
chromosome at the cell poles. The mitotic apparatus
disappears and chromosomes begin to lengthen as they
unwind. Cytokinesis, formation of a new cell membrane, occurs midway
between the daughter nuclei.
In plants, such as the onion root tip cells, this is seen as the formation of a
cell plate, dividing the original cell into two (presumably equivalent) daughter
cells. Cells now enter the G1, stage of interphase in the cell cycle and the
process begins anew.
Practical Genetics 1st ed. 2008
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Exercise 1
Mitotic Cell Division
Figure 1.3. The stages of mitosis in onion root tip cells.
In a growing plant root, the cells at the tip of the root are constantly dividing to
allow the root to grow. Because each cell divides independently of the others,
a root tip contains cells at different stages of the cell cycle. This makes a root
tip an excellent tissue to study the stages of cell division.
In this exercise, the student will prepare slides containing
stained onion root tip squash sections, which will allow him
to identify different stages of mitosis.
Procedure
Materials
Slides & cover slips
Microscope
Fresh onion root tips
Fixative ( methanol-acetic acid 3:1 v/v)
Forceps
1 M HCl
Razor blade
Stain
Paper towel, or absorbent paper
Caution
Fixative (3:1 methanol to
acetic acid) is a dangerous
and
volatile
solution.
Methanol is a flammable
poison capable of causing
skin irritation, blindness, CNS
depression and death. Acetic
acid is a flammable liquid
capable of causing severe
eye, skin, and respiratory
chemical burns. Always wear
personal protective equipment, wash with plenty of water
and move to fresh air if
exposed.
Method
Root tip preparation:
1. Obtain two small cups, label one of them with " HCl " and pour
enough 1 M HCl into it to cover the bottom, likewise, label the
other " fixative " and pour enough fixative fluid in it to cover the
bottom.
Practical Genetics 1st ed. 2008
5
Exercise 1
Mitotic Cell Division
2. Use forceps to transfer an onion root tip into the cup of HCl. After
4 minutes, transfer the root tip in the fixative and leave it for 4
minutes. Then place the root tip on a slide.
3. With a razor blade, or other sharp instrument cut off one to two
mm of the root tip and discard all except the tip that you want to
prepare.
4. Cover the root tip with a few drops of stain for 2 minutes, then blot
away the stain. Be careful not to touch the root tip!
5. Cover the root tip with one to two drops of water put a cover slip
over the root, put a paper towel or other absorbent paper and with
your thumb firmly press on the cover slip.
Note: Do not twist the cover slip! This pressure will spread the cells
into a single layer.
6. Observe your preparation under the low power
(X10) of a microscope (Figure 1.4).
7. Search the slide to find cells in various stages
of cell division, once you have located cells in
division, change to high power (X40) & try to
observe several stages of division.
8. Record the number of cells in each stage.
Count at least three full fields of view. You
should have counted over 200 cells.
9. Record your data in the table below.
1. Calculate the percentage of cells in each
phase and record in the table below.
Figure 1.4. Longitudinal section of
onion root tip.
Number of
cells
Percentage of total
cells counted
Interphase
Prophase
Metaphase
Anaphase
Telophase
Total
Calculate the Mitotic Index
Mitotic Index
Practical Genetics 1st ed. 2008
=
Number of cells in mitotic phase
Total number of cells counted
6
2
Exercise 2: Agarose Gel Electrophoresis
Objectives
•
•
To understand the principle of Gel electrophoresis.
To become familiar with the part of the electrophoresis setup.
Introduction
Electrophoresis is a laboratory technique for separating molecules based on
their charge. Molecules with a negative charge (anions) will be attracted to the
positively charged node (anode). Molecules with a positive charge (cations)
will be attracted to the negatively charged node (cathode).
Agarose gel electrophoresis is a widely used procedure in various areas of
biotechnology. This simple, but precise, analytical procedure is used in
research, biomedical and forensic laboratories. Of the various types of
electrophoresis, agarose gel electrophoresis is one of the most common and
widely used methods. It is a powerful separation method frequently used to
analyze DNA fragments, and it is a convenient analytical method for
determining the size of DNA molecules in the range of 500 to 30,000 base
pairs. It can also be used to separate other charged biomolecules such as
dyes, RNA and proteins.
The separation medium is a gel made from agarose, which is a
polysaccharide derivative of agar. Originating from seaweed, agarose is highly
purified to remove impurities and charge. It is derived from the same seaweed
as bacterial agar used in microbiology, as well as a food product called agaragar.
How Separation Occurs
The degree of separation and rate of molecular migration of molecules in a
mixture depends upon two main factors, the electrical charge and the size of
molecules (Figure 2.1).
Positive Molecules
Analyze
Charge
Separation
Size
Separation
Identify
Purify
Mixture of
Charged Molecules
Negative Molecules
Figure 2.1. Mixture of charged molecules can be separated into positive and negative ions
due to the electric field, then they are separated according to their size due to gel.
Practical Genetics 1st ed. 2008
Exercise 2
Agarose Gel Electrophoresis
1- The electrical charge
In electrophoresis, the electric charge often is passed through what is known
as a support medium. In general, the medium is mixed with a chemical
mixture called a buffer. The buffer carries the electric charge that is applied to
the system. The medium/buffer matrix is placed in a tray. Samples of
molecules to be separated are loaded into wells or slots that have been
formed at one end of the matrix. As electrical current is applied to the tray, the
matrix takes on this charge and develops positively and negatively charged
ends. As a result, molecules that are negatively charged such as
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are pulled toward the
positive end of the gel.
Proteins have net charges determined by charged groups of the amino acids
from which they are constructed. Proteins can also be amphoteric compounds
(a compound that can take on a negative or positive charge depending on the
surrounding conditions). A protein in one solution might carry a positive
charge in a particular medium and thus migrate toward the negative end of the
matrix. In another solution the same protein might carry a negative charge and
migrate toward the positive end of the matrix. For each protein there is a pH in
which the protein molecule has no net charge (the isoelectric point).
2- Size of Molecules
The agarose gel consists of microscopic pores that act as a molecular sieve
which separates molecules based upon size and shape. The charge to mass
ratio is the same for different sized DNA molecules. Therefore, the absolute
amount of charge on the molecule is not a critical factor in the separation
process.
The separation occurs because smaller molecules pass through the pores of
the gel more easily than larger ones (Figure 2.2), i.e., the gel is sensitive to
the physical size of the molecule. If the size of two fragments are similar or
identical, they will migrate together in the gel.
Porous
Material
Molecules
Entering Porous
of agarose
gel representing
Material
Smallest Move
Fastest
Figure 2.2. Crosssectional area
the pores present, small
molecules move faster than large molecules. Photo at the left represents a scanning electron
micrograph of agarose gel.
Molecules can have the same molecular weight and charge but different
shapes, as in the case of plasmid DNAs. Molecules having a more compact
shape (a sphere is more compact than a rod) can move more easily through
the pores. The migration rate of linear fragments of DNA is inversely
Practical Genetics 1st ed. 2008
8
Exercise 2
Agarose Gel Electrophoresis
proportional to the log10 of their size in base pairs. This means that the
smaller the linear fragment, the faster it migrates through the gel. In addition,
different molecules can interact with agarose to varying degrees. Molecules
that bind more strongly to the agarose will migrate more slowly. The mobility
of molecules during electrophoresis is also influenced by gel concentration.
Higher percentage gels, as well as thicker gels, are sturdier and easier to
handle. However, the mobility of molecules will take longer because of the
tighter matrix of the gel (Figure 2.3).
B
A
Figure 2.3. "A" is a 1% agarose gel, the pores are bigger than "B" gel which is a 2% agarose
gel.
In this exercise we will electrophorese various dyes which will approximate the
physical properties of the negatively charged biomolecules ( eg. DNA, RNA
and many proteins).
Procedure
Materials
• Horizontal gel electrophoresis apparatus
• Agarose
• Electrophoresis buffer
• Direct Current (D.C.) power supply
• Automatic micropipettes with tips
• Balance
• Microwave or hot plate
• 250 ml flasks or beakers
• Distilled or deionized water
Electrophoresis Buffers
Depending on the size of the DNA
electrophoresed and the application, different buffers can be used for agarose
electrophoresis. TAE buffer (Tris Acetate
EDTA) is the most common used
agarose gel electrophoresis buffer. TAE
has the lowest buffering capacity of the
buffers, however TAE offers the best
resolution for larger DNA. However, TAE
requires a lower voltage and more time.
However TBE buffer (Tris/Borate/EDTA)
is often used for smaller DNA fragments
(ie less than 500bp).
Method
PREPARING THE GEL BED
1. Close off the open ends of a clean and dry gel bed
(casting tray) by using rubber dams. Place a rubber dam
on each end of the bed. Make sure the rubber dam fits
firmly in contact with the sides and bottom of the bed.
2. Place a well-former template (comb) in the first set of
notches at the end of the bed. Make sure the comb sits
firmly and evenly across the bed.
Practical Genetics 1st ed. 2008
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9
Exercise 2
Agarose Gel Electrophoresis
CASTING AGAROSE GELS
3. Weigh the specified amount of agarose and transfer it to
a 250 ml flask.
4. Add the specified amount of buffer, swirl the mixture to
disperse clumps of agarose powder.
5. Heat the mixture to dissolve the agarose powder. The
final solution should appear clear (like water) without
any undissolved particles.
Microwave method:
Heat the mixture on High for 1 minute. Swirl the mixture
and heat on High in bursts of 25 seconds until all the
agarose is completely dissolved.
6. Cool the agarose solution to 55°C with careful swirling to
promote even dissipation of heat.
7. Pour the cooled agarose solution into the bed. Make
sure the bed is on a level surface. Allow the gel to
completely solidify. It will become firm and cool after
approximately 20 minutes.
1
2
3
Preparing the gel for electrophoresis
8. After the gel is completely solidified, carefully and
slowly remove the rubber dams from the gel bed. Be
especially careful not to damage or tear the gel wells
when removing the rubber dams.
9. Remove the comb by slowly pulling straight up. Do this
carefully and evenly to prevent tearing the sample
wells.
10. Place the gel (on its bed) into the electrophoresis
chamber, properly oriented, centered and level on the
platform.
11. Fill the electrophoresis apparatus chamber with the
required volume of diluted buffer.
Loading the samples
12. Load each of the dye samples (A – E) into the wells in
consecutive order. The amount of sample that should be
loaded is 10 µl.
Running the gel
13. After the samples are loaded, carefully put the cover
down onto the electrode terminals. Make sure that the
negative and positive color-coded indicators on the
cover and apparatus chamber are properly oriented.
14. Insert the plug of the black wire into the black input of
the power source (negative input). Insert the plug of the
red wire into the red input of the power source (positive
input).
15. Set the power source at the required voltage and
conduct electrophoresis for the length of time
Practical Genetics 1st ed. 2008
4
5
6
7
9
10
Exercise 2
Agarose Gel Electrophoresis
determined by your instructor. General guidelines are
presented in Table 1.
16. Check to see that current is flowing properly - you
should see bubbles forming on the two platinum
electrodes.
17. After approximately 10 minutes, you will begin to see
separation of the colored dyes. After the electrophoresis
is completed, turn off the power, unplug the power
source, disconnect the leads and remove the cover.
18. Document the gel results. A variety of documentation
methods can be used, including drawing a picture of the
gel, taking a photograph, or scanning an image of the
gel on a flatbed scanner.
12
13
Calculation and Analysis
Measure the distance that each of the color bands migrated
from the origin in the wells and record results in the table
below. Rate of migration of a molecule is inversely
proportional to the log of its molecular weight
Distance α 1 / log10-MW
Construct a standard curve to relate the distance of migration of each dye to
its assumed molecular weight. From this standard curve estimate the
molecular weight of the unknown dye.
Dye
Cresol red
Bromophenol blue
Eosin
Orange G
Trypan blue
Practical Genetics 1st ed. 2008
Molecular Weight/ Dalton
382.42
669.96
691.85
Distance migrated/ mm
960.81
11
Agarose Gel Electrophoresis
Log- Molecular weight
Exercise 2
Distance/ mm
Practical Genetics 1st ed. 2008
12
3
Exercise 3: Human Metaphase Chromosomes
Objectives
Preparing, staining and observing human metaphase chromosomes.
Introduction
Chromosome Morphology
Chromosomes are not visible under the light microscope in non-dividing
(interphase) cells. As the cell begins to divide, the threads of chromatin (DNAprotein complex) in the nucleus begin to condense into multiple levels of
coiled structures recognizable as chromosomes. There are two modes of cell
division: mitosis and meiosis. Because mitotic cells are easy to obtain,
morphological studies are generally based on mitotic metaphase
chromosomes.
Chromosome Structure
At metaphase the chromosomes are at their most condensed state, with
spindle fibers attaching to the area of the centromere called the kinetochore
(Figure 3.1). Anaphase begins with the division of the centromere and the
separation of chromatids. Once separated, each chromatid is known as a
chromosome. The best mitotic stage for chromosome analysis is prometaphase or metaphase. A typical metaphase chromosome consists of two arms
separated by a primary constriction or centromere.
Each of the two sister-chromatids contains a highly
coiled double helix of DNA. Often the sister chromatids are so close to each other that the whole
chromosome appears as a single rod-like structure.
A chromosome may be characterized by its total
length and the position of its centromere (Figure
3.2).
Chromosome Number
The diploid chromosome number is the number of
chromosomes in the somatic cell and is designated
by the symbol 2N. The gametes, which have onehalf the diploid number, have the haploid number N.
Figure
3.1.
Chromosomes
are
In humans the diploid number is 46, with 23 inhereplicated during S phase, before
rited from each parent through the sperm or egg.
mitosis begins. Two genetically identical
chromatids of a replicated chromosome
Same (homologous) chromosomes form a pair with
join at the centromere at the
one member from each parent. Thus, there are 23
kinetochore area (a). In the photograph
pairs of chromosomes in human cells. Of these, 22
(b), a human chromosome is in the
pairs are not directly involved in sex determination,
midst of forming two chromatids.
and are known as autosomes. The remaining
chromosome pair consists of the sex chromosomes, and is directly involved in
sex determination. In females the two sex chromosomes are identical (XX),
Practical Genetics 1st ed. 2008
Exercise 3
Human Metaphase Chromosome
whereas in males the two sex chromosomes are
not identical (XY).
Cytogenetics, the study of chromosomes,
originated more than a century ago. However,
not until the last 30 years or so have human
chromosome studies become a major field in the
biomedical sciences. Chromosome banding
methods, for example, are today’s vital tools in
clinical genetics and evolutionary studies.
Furthermore, human cytogenetics coupled with
molecular techniques has revolutionized the field
of molecular genetics, including gene mapping
and recombinant DNA technology.
Figure 3.2. Chromosome morphology and
terminology.
Specimen
In order to successfully culture blood samples for cytogenetic analysis they
have to be received fresh and unclotted. Heparinized whole blood (green top
vacutainer tube); sodium heparin is the recommended anticoagulant. Any
other choice of tube is unsuitable since the sample will either arrive clotted or
tube will contain EDTA which is toxic to cells and will therefore adversely
affect the culture.
Sterile Technique
Aseptic or sterile technique is the performance of tissue culture procedures
without introducing contaminating micro-organisms from the environment. In
doing tissue culture work, 70% of the problems are due to a lack of good
sterile technique. Microorganisms causing the contamination problems exist
everywhere, on the surface of all objects and in the air. Tissue culture media
used are often supplemented with antibiotics. Antibiotics do not eliminate
problems of gross contamination which result from poor sterile technique or
antibiotic-resistant mutants. Autoclaving renders pipettes, glassware, and
solutions sterile.
Overview of procedure for chromosome preparation
A variety of tissue types can be used to obtain chromosome preparations.
Some examples include peripheral blood, bone marrow, amniotic fluid, skin
fibroblasts and products of conception. In the case of blood cell culture only
cells that are actively dividing can be used for cytogenetic studies. Normally
only white blood cells are used for cytogenetic analysis (i.e. neutrophils,
eosinophils, basophils, monocytes and lymphoctes). These cells contain a
nucleus and are capable of undergoing cell division. Although specific
techniques differ according to the type of tissue used, the basic method for
obtaining chromosome preparations is as follows:
1- Cell culture: although cell culture methods vary significantly with the tissue
of origin (amniotic fluid, chorionic villi, fetal tissues, peripheral blood, bone
marrow, solid tumors, cell lines of various origins), the final goal is to achieve
cell growth and division, ultimately leading to a good mitotic index.
Practical Genetics 1st ed. 2008
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Exercise 3
Human Metaphase Chromosome
2- Harvesting: mitotic spindle formation is blocked,
usually by adding colcemide to the culture, and the cell
division is stopped at the metaphase level. Cells are
subjected to hypotonic treatment, which increases their
volume, and disrupts the cell membrane of the red blood
cells allowing their removal. A fixative solution is added to
the cell suspension to preserve the cells in their "swollen"
state and to remove the water, thus "hardening" the
biologic
material.
The
common
fixative
(3:1
methanol:acetic acid) removes lipids and alters/denatures
proteins thus making the cell membrane remnant very
fragile, which is important for subsequent chromosome
spreading.
3- Slide preparation: drops of cell suspension are placed
on a slide, and allowed to dry in a controlled fashion,
leading to chromosome spreading.
Caution
Fixative (3:1 methanol to
acetic acid) is a dangerous
and
volatile
solution.
Methanol is a flammable
poison capable of causing
skin irritation, blindness, CNS
depression and death. Acetic
acid is a flammable liquid
capable of causing severe
eye, skin, and respiratory
chemical burns. Always wear
personal
protective
equipment, wash with plenty
of water and move to fresh air
if exposed.
4- Staining: stain chromosome preparations to detect possible numerical and
structural changes.
Procedure
Materials
Pre-cleaned microscope slides
5 ml peripheral blood in sodium heparin (500 U/ml)
0.04 mg/ml colchicine
Lymphocyte culture medium: RPMI 1640, 100000 U/ml penicillin, 100
mg/ml streptomycin, 20% fetal bovine serum (FCS), Phytohemagglutinin 20µg/ml
Hypotonic solution: 0.075 M KCl
Fixative: 3:1 methanol/ acetic acid at 4°C
Method
Cell culture
1. Label a 15 ml sterile culture tube, pipette 10 ml RPMI 1640 medium with LGlutamine. Add the following supplements to the tube: 10 µl Penicillinstreptomycin, 0.3 ml Phytohemagglutinin and 2 ml of Fetal bovine serum.
2. Add 1 ml of heparinized blood into the medium tube.
3. Mix the contents of the tube with gentle inversion, then incubate the tube in
5% CO2 incubator at 37°C for 72 hours in a slant position.
Harvesting
a- Stopping cell division at metaphase
4. After 72 hr culture, add 25 µl of pre-warmed colchicine (37C) and mix well
gently. Then incubate the tube for 30 min at 37°C.
b- Hypotonic treatment of cells
5. Centrifuge tubes at 2000 rpm for 10 min.
Practical Genetics 1st ed. 2008
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Exercise 3
Human Metaphase Chromosome
6. Discard supernatant without disturbing the cells on the bottom
leaving about 0.5 ml of medium above the cell pellet.
7. Resuspend the cells in the remaining medium and carefully add
1 ml of prewarmed (37°C) 0.075 M KCl, drop-by-drop, while
agitating gently. Add an additional 9 ml of KCl, for a total of 10
ml; mix well.
8. Incubate for 15 min at 37°C in the incubator.
Important Note
Hypotonic
solution
should not remain in
contact with the cells
for more than 27
minutes.
Excess
exposure may cause
rupture of WBCs.
c- Fixation of Cells
9. Centrifuge tube at 1500 rpm for 10 minutes.
10. Remove supernatant leaving about 0.5 ml of fluid above the cells.
11. Add 5 ml of freshly prepared fixative, the first 2 ml should be added
dropwise while agitating gently, recap the tube and invert to mix.
12. Incubate the tube in the refrigerator for 30 minutes.
13. Centrifuge the tube at 1500 rpm for 10 minutes.
14. Remove the supernatant and add 6 ml of fixative, mix well and then
centrifuge at 1500 rpm for 10 minutes.
15. Repeat step 14 one or two times as needed.
16. Remove supernatant leaving about 1 ml of fixative, resuspend cells. This
is the material which is going to be used for slide preparation.
d- Slide Preparation and Staining
The slides should be exceptionally clean.
17. Withdraw a few drops of the cell suspension with a Pasteur pipette.
18. From a height of approximately 20 cm drop 2 or 3 drops of fluid onto each.
slide.
19. Allow the slides to dry, the best way is to place the slide in an incubator at
37°C overnight
20. Stain the slides by immersion in freshly prepared Giemsa stain for 7-10
minutes.
21. Remove slides from stain and rinse in distilled water until all excess satin
is removed.
e- Observing human metaphase chromosomes
Observe the slides under the microscope, first locate the field under low
power (X10) lens, then observe the slide under high power (X40) lens and
determine the best metaphase spread. Now observe the slide under oil
immersion lens, and start examination of different chromosomes.
a
b
Figure 3.3. "a" represents a metaphase spread under high power lens,
"b" represents a metaphase spread under oil immersion lens.
Practical Genetics 1st ed. 2008
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4
Exercise 4:
Human Chromosome Identification by G-Banding (karyotyping)
Objectives
•
•
Preparation, Staining and Observing G-banded human chromosomes.
Develop an understanding of karyotyping and the association of
various chromosomal abnormalities to diseases.
Introduction
Chromosomes are composed of double-stranded DNA associated with
specific proteins. The nuclei of normal human somatic cells each contain 23
pairs of chromosomes, one of each pair derived from either maternal or
paternal origin. During metaphase, chromosomes become condensed and
stain intensely with basic dyes. The size and staining patterns allow the
differentiation of different chromosomes. The autosomes, or nonsex
chromosomes, are numbered from 1 to 22 approximating decreasing size
order. The 23rd pair is the sex chromosomes, X and Y; normal females
contain two X chromosomes per nucleus, whereas normal males contain one
X and one Y chromosome. Variations in the karyotype, can be used to
diagnose certain disorders (e.g. Down syndrome), prenatal abnormalities or
certain types of tumors.
A karyotype is a display or photomicrograph of an
individual’s somatic-cell metaphase chromosomes
that are arranged in a standard sequence, usually
based on number, size, and type (Figure 4.1).
Chromosome abnormalities
Chromosome abnormalities can be numerical, as in
the presence of extra or missing chromosomes, or
structural as in translocations, inversions, large scale
deletions or duplications. Numerical abnormalities,
also known as aneuploidy, often occur as a result of
nondisjunction during meiosis in the formation of a
gamete; trisomies, in which three copies of
chromosome are present instead of the usual two,
are common numerical abnormalities.
Some chromosomal abnormalities that lead to
disease in humans include:
•
•
•
Figure 4.1. Karyotype of a normal male.
Turner syndrome results from a single X chromosome (45, X or 45, X0).
Klinefelter syndrome, the most common male chromosomal disease,
otherwise known as 47, XXY is caused by an extra X chromosome.
Down syndrome, a common chromosomal disease, is caused by trisomy
of chromosome 21.
Some disorders arise from loss of just a piece of one chromosome, including:
Practical Genetics 1st ed. 2008
Exercise 4
•
•
•
Human Chromosome Identification by G-Banding
Cri du chat (cry of the cat), from a truncated short arm on chromosome
5. The name comes from the babies' distinctive cry, caused by
abnormal formation of the larynx.
1p36 Deletion syndrome, from the loss of part of the short arm of
chromosome 1.
Angelman syndrome – 50% of cases have a segment of the long arm
of chromosome 15 missing.
Chromosomal abnormalities can also occur in cancerous cells of an otherwise
genetically normal individual; one well-documented example is the
Philadelphia chromosome, a translocation mutation commonly associated with
chronic myeloid leukemia and less often with acute
lymphoblastic leukemia.
Identification of Chromosomes
The pairs of chromosomes are differentiated according to
the following characteristics (Figure 4.2):
Size: This is the easiest way to tell two different
chromosomes apart.
Banding pattern: The size and location of Giemsa
bands on chromosomes make each chromosome
pair unique.
Centromere position: Centromeres are regions in
chromosomes that appear as a constriction.
Using these key features, scientists match up the 23 pairs.
Figure 4.2. key features to identify
chromosome similarities and differrences.
Performing a Karyotype
The slides are scanned for metaphase spreads and
usually 10 to 30 cells are analyzed under the microscope by a cytogeneticist.
When a good spread (minimum number of overlapping chromosomes) is
found, a photograph is taken or the analysis is done by a computer. The
chromosomes are arranged in a standard presentation format of longest to
shortest. Actually chromosome 21 is smaller than chromosome 22, however,
since Trisomy 21 (Down Syndrome) had already been named, it was decided
to leave the numbering system as it was.
Centromere position
The centromere is the location of spindle attachment and is an integral part of
the chromosome. It is essential for the normal movement and segregation of
chromosomes during cell division. Human metaphase chromosomes come in
three basic shapes and can be categorized according to the length of the
short and long arms and also the centromere location (Figure 4.3).
Metacentric chromosomes have short and long arms of roughly equal length
with the centromere in the middle. Submetacentric chromosomes have short
and long arms of unequal length with the centromere more towards one end.
Acrocentric chromosomes have a centromere very near to one end and have
very small short arms. They frequently have secondary constrictions on the
short arms that connect very small pieces of DNA, called stalks and satellites,
to the centromere.
Practical Genetics 1st ed. 2008
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Exercise 4
Human Chromosome Identification by G-Banding
Figure 4.3. Classification of chromosomes according to the centromere position.
Banding patterns
Special chromosome-staining procedures have revealed specific sets of
intricate bands (transverse stripes) in many different organisms. The positions
and sizes of the bands are highly chromosome specific. One of the basic
chromosomal banding patterns is that produced by Giemsa reagent, a DNA
stain applied after mild proteolytic digestion of the chromosomes. This reagent
produces patterns of light-staining (G-light) regions and dark-staining (G-dark)
regions. The patterns are consistent within species. In the complete set of 23
human chromosomes, there are approximately 550 G-dark bands visible at
metaphase of mitosis. These bands have provided a useful way of subdividing
the various regions of chromosomes, and each band has been assigned a
specific number.
The difference between dark- and light-staining regions was believed to be
caused by differences in the relative proportions of bases: the G-light bands
being relatively GC-rich, and the G-dark bands AT-rich. However, it is now
thought that the differences are too small to account for banding patterns. The
crucial factor appears to be chromatin packing density: the G-dark regions are
packed more densely, with tighter coils, which results in a higher density of
DNA to take up the stain.
In addition, various other correlations have been made. For example,
deoxynucleotide-labeling studies showed that G-light bands are early
replicating. Furthermore, if polysomal (polyribosomal) mRNA (representing
genes being actively transcribed) is used to label chromosomes in situ, then
most label binds to the G-light regions, suggesting that these regions contain
most of the active genes. From such an analysis, it was presumed that the
density of active genes is higher in the G-light bands.
Practical Genetics 1st ed. 2008
19
Exercise 4
Human Chromosome Identification by G-Banding
Procedure
Materials
Hank’s Balanced Salt Solution (HBSS)
Giemsa stain
Phosphate Buffer, pH 6.8
Normal saline (0.9%)
0.25% Trypsin (working solution is prepared by mixing 5 ml of 0.25%
Trypsin with 45 ml normal saline)
Method
We are going to follow the same procedure indicated in exercise 3 till we
prepare the slides, and they will be ready for staining. Method for obtaining
chromosome preparations is as follows:
• Cell culture,
• Harvesting,
• And Slide preparation.
Age slides
Place fixed dry slides on slide rack in 95oC oven and bake for 20 minutes.
Take slides out of the oven and leave them to cool.
Trypsinize slides with metaphase chromosomes
Immerse aged slides in working Trypsin Banding Solution at room
temperature for 15-120 seconds with a forceps.
Follow the Trypsin treatment with a brief immersion and swirl in phosphate
buffer in a coplin jar to stop the action of the Trypsin.
Stain slides in Giemsa stain
Immerse the Trypsinized slides in working Giemsa stain for 2 minutes.
Rinse slides thoroughly with distilled water.
Tap off excess water on a paper towel, wipe the back of the slide dry and
leave the slide to dry.
Observe the slides under the microscope, first locate the field under low
power (X10) lens, then observe the slide under high power (X40) lens and
determine the best metaphase spread. Now observe the slide under oil
immersion lens, and start identification of different chromosomes.
Procedure notes
Time of trypsin treatment varies with each case and is dependent on the
age of the slides to be banded, the technique used to make the slides,
and the temperature and concentration of the trypsin solution.
Chromosomes are under-trypsinized if they stain homogenously dark,
banding is present but the bands appear to blur into each other, or if
banding is present but a darkly stained bar is noticeable between the
chromatids.
Chromosomes are over-trypsinized if they appear swollen, fuzzy, or have
a cobwebbed appearance.
Practical Genetics 1st ed. 2008
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Exercise 4
Human Chromosome Identification by G-Banding
Due to variation in the stages of condensation of chromosomes on the
same slide, some spreads will be over-trypsinized and some will be
under-trypsined. The goal is to maximize the number of analyzable
metaphases for a given slide.
It is important to mix the working Leishman’s stain fresh before using,
and change the solution if it has been sitting for more than approximately
30 minutes.
Slides that are under-Trypsinized may be destained in fixative (3:1
methanol to acetic acid), rinsed in water, re-Trypsinized for a few
seconds, and then re-stained.
Cut and Paste
Cut the chromosomes present on page number 23 and paste each
chromosome near its homologous pair on page 22. Now, indicate if the
karyotype is normal or abnormal, if abnormal; indicate the abnormality present
Practical Genetics 1st ed. 2008
21
Exercise 4
Human Chromosome Identification by G-Banding
X or Y
Practical Genetics 1st ed. 2008
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Exercise 4
Practical Genetics 1st ed. 2008
Human Chromosome Identification by G-Banding
23
5
Exercise 5: Extraction of Human DNA
Objectives
• Isolation of genomic DNA from human blood.
• Analysis of isolated DNA using agarose gel electrophoresis and
spectrophotometery.
Deoxyribonucleic Acid (DNA)
A nucleic acid that carries the genetic information in the cell and is capable of
self-replication and synthesis of RNA. DNA consists of two long chains of
nucleotides twisted into a double helix and joined by hydrogen bonds between
the complementary bases adenine and thymine or cytosine and guanine
(Figure 5.1). The sequence of nucleotides determines individual hereditary
characteristics.
DNA plays an important role in two processes. During the process of
replication, DNA provides information to copy itself, so genetic information can
be passed on from generation to generation of cells. DNA also provides
instructions for making proteins, which are vital to the maintenance and
function of cells. DNA provides information for the
order of amino acids required for making various
proteins. A large number of proteins and enzymes
including DNA polymerases are involved in the
synthesis of DNA.
The human genome consists of about 2.9 billion
base pairs. Of this total, only about 5% code for
protein. Intervening sequences and other
noncoding sequences make up the remainder.
Some of the noncoding sequences or other
functionally unassigned sequences may possess
undiscovered functions. In addition to genomic
DNA, mitochondria, which are cellular organelles,
contain their own DNA (mitochondrial DNA) which
replicate independently from cell chromosomal
DNA.
Figure 5.1. The DNA double helix.
When cells are chemically lysed (broken open),
DNA from chromosomes is released and can be isolated and purified. DNA
extraction is frequently the first step for molecular biology and biotechnology
experiments. Extracted DNA is soluble in water and thus will appear as a clear
solution. By contrast DNA is insoluble in salt solutions and alcohol, where it
will form white fibers. Purification procedures for DNA usually include
precipitation with alcohol in the presence of salt. A glass rod or a stirrer is
used to spool DNA and to separate the DNA from the solution. The DNA will
appear as a viscous, clotted mass. The amount of DNA spooled will vary and
is a consequence of the intactness of the DNA sample.
Practical Genetics 1st ed. 2008
Exercise 5
Extraction of Human DNA
Almost any tissue or body fluid may be used as a source of DNA. The most
common sources of human DNA are samples from hair, cheek cells, blood
and saliva. Once extracted, DNA can be stored for long periods of time.
Various methods of storage include precipitation and storage under alcohol at
room temperature or under refrigeration. DNA can be recovered from small
blood spots and tissues, or even a few cells. Such amounts of DNA can be
obtained from individuals during medical procedures or evidence left behind in
crime scenes such as cells that are recovered from the fingernails of a victim.
In recent times, a few cells deposited by a person while licking and sealing an
envelope has been sufficient to obtain DNA and match the DNA fingerprint to
the person who left this evidence behind.
There are three basic steps in a DNA extraction, the details of which may vary
depending on the type of sample and any substance that may interfere with
the extraction and subsequent analysis.
• Break open cells and remove membrane lipids.
• Remove cellular and histone proteins bound to the DNA, by adding a
protease, by precipitation with sodium or ammonium acetate, or by
using a phenol/chloroform extraction step.
• Precipitate DNA in cold ethanol or isopropanol, DNA is insoluble in
alcohol and clings together, this step also removes salts.
Analysis of the isolated DNA
The product of DNA extraction will be used in subsequent experiments. Poor
quality DNA will not perform well in PCR. Quality and quantity of the extracted
DNA can be analyzed by 2 methods:
• agarose gel electrophoresis, where a good quality DNA is represented
by a sharp band near the wells of the gel, while smearing indicates
DNA degradation (Figure 5.2),
• and spectrophotometery for quantitation and purity.
Figure 5.2. A Photograph representing the quality of 16 DNA samples extracted from whole
blood samples, run on ethidium bromide stained 1% agarose gel. Lanes 1,3,5,7,9,11,13 & 15
represent good quality DNA, while lanes 2, 4, 6, 8, 10, 12, 14 & 16 represent bad quality
(broken) DNA.
Practical Genetics 1st ed. 2008
25
Exercise 5
Extraction of Human DNA
Procedure
Blood samples should be collected in disodium EDTA tube. Samples can be
stored at -20oC or -70oC. Fresh samples are kept in freezer for a few hours to
facilitate RBCs hemolysis. Allow samples to thaw before starting the
extraction.
Materials:
• Erythrocyte lysing buffer (0.155M NH4Cl, 10 mM KHCO3, 0.1 mM
Na2EDTA, pH 7.4)
• SE buffer (75 mM NaCl, 25 mM Na2EDTA, pH 8.0) containing 100
µg/ml of Proteinase K & 1% sodium dodecyl sulphate (SDS)
• 6 M NaCl
• Chloroform
• Isopropanol
• 75% ethanol
• TE buffer (1 M Tris-HCl; 0.5 M EDTA; pH 8.0)
1. Lysis of red blood cells
• Pipette 3 mls of whole blood in a conical centrifuge tube
• Add 9 mls of 1X erythrocyte lysing buffer
• Leave 10 minutes at RT, mix occasionally
• Centrifuge at 4000 rpm for 5 min
• Discard supernatent
• White pellet is observed at bottom of tube
• Wash pellet 3 times by adding 3 mls of buffer, incubate 10 min at RT, &
centrifuge
2- Lysis of leukocytes and leukocytes' nuclei
• Add 1.5 mls of SE buffer to the pellet
• Incubate at 37-55oC overnight in a water bath or incubator
• WBCs denatured, nuclei are lysed & DNA goes out in solution
3- Extraction of proteins
•
•
•
•
•
After the incubation, add 1.5 ml of SE buffer together with 750 µl of 6 M
NaCl and then add 3.75 ml chloroform.
The tubes are mixed vigorously (on vortex) for 20 seconds with
occasional mixing for at least 30 minutes
Centrifuge for 10 minutes at 2000 rpm with minimal breaking force
After centrifugation 2 phases are observed and care must be taken not
to disturb the interphase
The upper phase contains the DNA while proteins are in the lower
phase
4- DNA precipitation and wash
• The upper phase containing the DNA is transferred to a clean and
sterile conical centrifuge tube
Practical Genetics 1st ed. 2008
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Exercise 5
•
•
•
•
•
•
•
Extraction of Human DNA
Add an equal volume of isopropanol
DNA will be precipitated by gentle swirling & observed as a white
thread like strand
Using a sterile spatula or loop transfer the DNA strand into a sterile
microcentrifuge tube containing 1 ml of 75% ethanol
Wash DNA by inversion to remove any remaining salts
Centrifuge at 11000 g for 4 minutes, then discard supernatant taking
care not to discard the pellet
Repeat the washing step, then centrifuge
Remove supernatant, and dry the pellet by using a vacuum centrifuge
or by leaving the tubes opened and inverted in an oven at 50-65oC for
1 hour
5- Resuspension of DNA
•
Dried pellet is resuspended in TE buffer and left overnight on a rotator
6- Check quantity and quality DNA
Quality and quantity of the extracted DNA can be analyzed by 2 methods:
1- Agarose gel electrophoesis
Prepare agarose gel of 0.6% to 1% concentration, mix DNA samples with
loading buffer and then load on the gel. Electrophorese at 70–80 volts, for 45–
90 minutes. After electrophoresis stain the gel with ethidium bromide and then
view on UV transilluminator. A good quality DNA is represented by a sharp
band near the wells of the gel, while smearing indicates DNA degradation.
2- Spectrophotometry
Spectrophotometer is used to measure the quantity and purity of the extracted
DNA.
Quantity: Nucleic acids have a peak absorbance in the ultraviolet range at
about 260 nm
1 A260 O.D. unit for dsDNA = 50 µg/ml
DNA purity: The purity of the DNA is reflected in the OD260:OD 280 ratio and
must be between 1.7 and 2.00. Decreased 260:280 ratio means that too much
protein or other contaminant is present.
Practical Genetics 1st ed. 2008
27
6
Exercise 6: Polymerase Chain Reaction (PCR)
Objectives
• To understand how the PCR technique works
• To perform the PCR experiment
• To analyze the PCR products
Introduction
The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique
for DNA amplification in vitro. PCR amplifies specific DNA sequences
exponentially by using multiple cycles of a three-step process. PCR can
achieve more sensitive detection and higher levels of amplification of specific
sequences in less time than previously used methods, e.g. DNA cloning.
These features make the technique extremely useful, not only in basic
research, but also in commercial uses, including genetic identity testing,
forensics, industrial quality control and in vitro diagnostics. Basic PCR has
become commonplace in many molecular biology labs where it is used to
amplify DNA fragments and detect DNA or RNA sequences within a cell or
environment. However, PCR has evolved far beyond simple amplification and
detection, and many extensions of the original PCR method have been
described.
PCR is capable of amplifying sequences from minute amounts of target DNA,
even the DNA from a single cell. Such exquisite sensitivity has afforded new
methods of studying molecular pathogenesis and has found numerous
applications in forensic science, and in diagnosis, in genetic linkage analysis
using single-sperm typing. However, the extreme sensitivity of the method
means that great care has to be taken to avoid contamination of the sample
under investigation by external DNA, such as from minute amounts of cells
from the operator.
The cycling reactions
There are three major steps in a PCR (Figure 6.1),
which are repeated for 30 or 40 cycles. This is done on
an automated cycler, which can heat and cool the
tubes with the reaction mixture in a very short time.
Nucleic Acid Cross-Contamination
It is important to minimize crosscontamination between samples
and prevent carryover of RNA and
DNA from one experiment to the
next. Use separate work areas and
pipettes for pre- and postamplification steps. Use aerosolresistant tips to reduce crosscontamination
during
pipetting.
Wear gloves and change them
often.
1. Denaturation
The initial step denatures the target DNA by
heating it to 94°C or higher for 15 seconds to 2
minutes. In the denaturation process, the two
intertwined strands of DNA separate from one
another, producing the necessary single-stranded
DNA template for replication by the thermostable DNA polymerase.
2. Annealing
In this step the temperature is reduced to approximately 50–60°C. At this
temperature, the oligonucleotide primers can form stable associations
Practical Genetics 1st ed. 2008
Exercise 6
Polymerase Chain Reaction (PCR)
(anneal) with the denatured target DNA and serve as primers for the
DNA polymerase.
3. Extension
Finally, the synthesis of new DNA begins as the reaction temperature is
raised to the optimum for the DNA polymerase. For most thermostable
DNA polymerases, this temperature is in the range of 70–74°C. The
extension step lasts approximately 1–2 minutes. The next cycle begins
with a return to 94°C for denaturation.
Denaturation step: An initial denaturation step of 2–5 minutes at 94–95°C is
required prior to start PCR cycling to fully denature the DNA.
Figure 6.1. Schematic diagram of the PCR process.
PCR Reaction Components
Typical components of a PCR include:
DNA: the template used to synthesize new DNA strands. Use of high
quality, purified DNA templates greatly enhances the success of PCR
Practical Genetics 1st ed. 2008
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Exercise 6
Polymerase Chain Reaction (PCR)
reactions. It is also critical that contamination from outside sources,
especially previous PCR reactions, be avoided. Approximately 104
copies of the target DNA are required to detect a product in 25–30
cycles of PCR. Typically, this means a 1–10 µg/ml of genomic
templates.
DNA polymerase: an enzyme that synthesizes new DNA strands.
Taq DNA polymerase is isolated from Thermus aquaticus and
catalyzes the primer-dependent incorporation of nucleotides into
duplex DNA in the 5′→3′ direction in the presence of Mg2+. It is
recommended to use 1–1.25 units of Taq DNA polymerase in a 50µl
amplification reaction.
Two PCR primers: short DNA molecules (oligonucleotides) that
define the DNA sequence to be amplified. Oligonucleotide primers
are generally 20–30 nucleotides in length, and ideally have a GC
content of 40–60%, with GC residues spaced evenly within the
primer. Calculated melting temperatures (Tm) for the two primers
should be from 42–65°C, and the Tm for the two primers should be
within 5°C of each other.
Deoxynucleotide triphosphates (dNTPs): the building blocks for the
newly synthesized DNA strands. The final concentration of dNTPs is
typically 200 µM of each nucleotide.
Reaction buffer: a chemical solution that provides the optimal
environmental conditions. Most reaction buffers consist of a buffering
agent, most often a Tris-based buffer, and salt, commonly KCl. The
buffer regulates the pH of the reaction, which affects the DNA
polymerase activity and fidelity.
Magnesium: a necessary cofactor for DNA polymerase activity. A
magnesium concentration of 1.5–2.0 mM is optimal for most PCR
products generated with Taq DNA Polymerase. Optimization normally
involves supplementing the magnesium concentration in 0.5 or 1.0
mM increments.
Procedure
Materials
Standard Taq Reaction Buffer (10X)
Deoxynucleotide Solution Mix (10 mM)
Upstream Primer (10 µM stock)
Downstream Primer (10 µM stock)
DNA Template
Taq DNA Polymerase
Nuclease free water
Methods
In this experiment we are going to amplify part of the Human Growth Hormone
gene. Specific primers for this gene will to be used.
1. Prepare the master mix
When setting up multiple reactions it is faster and more accurate to create
a master mix of the components that are common to all reactions. In
Practical Genetics 1st ed. 2008
30
Exercise 6
Polymerase Chain Reaction (PCR)
general, this involves creating a stock solution of polymerase, nucleotides,
reaction buffer, water, and primers. The master mix is then aliquotted and
mixed with the DNA template.
PCR reaction mixture
Reagent
Buffer (X10)
MgCl2 (25 mM)
dNTPs (100mM)
Primer 1 (F)
Primer 2 (R)
Taq DNA polymerase
DNA template
Water
Total Volume
Volume
(µl)
2.0
1.6
Final
concentration
1X
2.0 mM
0.1
0.2
0.2
0.25
2.0
13.65
20
0.1 mM
1.0 µM
1.0 µM
2.0 U
100 ng
2- Program the thermocycler
The thermocycler profile for amplifying human growth
hormone gene is:
Step 1: Denaturation for 3 min. at 95oC
Melting for 60 sec. at 95oC
Annealing for 60 sec. at 57oC
Extension for 90 sec. at 72oC
Step 3: Final elongation for 10 min. at 72oC
Step 4: Hold at 4 oC
3- Run the samples on thermocycler
Place the samples in the thermocycler, close the lid
and start the program.
Volume X
No. of tubes
Important Note
Negative & positive control
reactions should be run with
patient samples.
Negative control to check for
contamination, contains all
reagents except the DNA
template.
Positive control to check if
PCR reaction has worked,
contains all reagents and a
known target-containing DNA
template.
Caution
4- Analysis of PCR products
Ethidium
bromide
is
a
Analyze products on 2% agarose gel containing
powerful mutagen and is
ethidium bromide (Ethidium Bromide, A fluorescent
toxic. Avoid breathing the
dye visualized when excited by UV light).
dust. Wear appropriate gloves
Loading Dye
when working with solutions
that contain this dye.
Samples are prepared with loading dye and then
loaded on the gel. It is used to prepare DNA markers and samples for
loading on agarose gels. It contains bromophenol blue dye, for visual
tracking of DNA migration during electrophoresis. The presence of
glycerol in the solution ensures that the sample sinks at the bottom of the
well.
Visualize the PCR product on UV transilluminator, There should be a 400 bp
band for the positive samples.
Practical Genetics 1st ed. 2008
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7
Exercixe 7:
Restriction Enzyme Digestion & Southern Blotting of DNA
Objectives
Digestion of DNA by a restriction enzyme.
Analysis of the digested DNA by electrophoresis.
Transfer the digested DNA to nitrocellulose membrane (Southern blotting)
Including the procedure of setting up the Southern blotting device.
Introduction
The discovery of restriction enzymes ushered in a new era of molecular genetics.
These enzymes cut the DNA molecule in a highly specific and reproducible way.
This, in turn, has lead to the development of molecular cloning and the mapping
of genetic structures.
Restriction enzymes are endonucleases which catalyze the cleavage of the
phosphodiester bonds within both strands of DNA. They require Mg+2 for activity
and generate a 5 prime (5') phosphate and a 3 prime (3') hydroxyl group at the
point of cleavage. The distinguishing feature of restriction enzymes is that they
only cut at very specific sequences of bases, Figure 7.1. Restriction enzymes are
obtained from many different species of bacteria. To date, over 3,000 restriction
enzymes have been discovered and catalogued.
Restriction enzyme is part of the cell’s
restriction-modification system in bacteria. The phenomenon of restriction modification in bacteria is a small scale
immune system for protection from
infection by foreign DNA. Bacteria can
protect themselves only after foreign DNA
has entered their cytoplasm. For this
protection, many bacteria specifically
mark their own DNA by methylating
bases on particular sequences with
modifying enzymes. DNA that is
Figure 7.1. The enzyme EcoRI cutting DNA at its
recognized as foreign by its lack of
recognition sequence.
methyl groups on these same sequences
is cleaved by the restriction enzymes and
then degraded by exonucleases to nucleotides.
Restriction enzymes are named according to the organism from which they are
isolated. This is done by using the first letter of the genus followed by the first two
letters of the species. Only certain strains or sub-strains of a particular species
may produce restriction enzymes. The type of strain or substrain sometimes
follows the species designation in the name. Finally, a Roman numeral is always
Practical Genetics 1st ed. 2008
Exercixe 7
Restriction Enzyme Digestion & Southern Blotting of DNA
used to designate one out of possibly several different restriction enzymes
produced by the same organism or by different substrains of the same strain.
A restriction enzyme requires a specific double stranded recognition sequence of
nucleotides to cut DNA. Recognition sites are usually 4 to 8 base pairs in length.
Cleavage occurs within or near the site. The cleavage positions are indicated by
arrows. Recognition sites are frequently symmetrical, i.e., both DNA strands in
the site have the same base sequence when read 5' to 3'. Such sequences are
called palindromes. Consider the recognition site and cleavage pattern of Eco RI
as an example.
As shown above, Eco RI causes cleavage of DNA. The ends of the DNA
fragments are called “sticky” or “cohesive” ends because the single-stranded
regions of the ends are complementary. Other restriction enzymes, such as
Hae III, introduce cuts that are opposite each other. This type of cleavage
generates “blunt” ends, as shown below.
In general, the longer the DNA molecule, the greater the probability that a given
recognition site will occur. Therefore, human chromosomal DNA, which contains
three billion base pairs, has many more recognition sites than a plasmid DNA
containing only several thousand base pairs.
Restriction enzymes are traditionally classified into three types on the basis of
subunit composition, cleavage position, sequence-specificity and cofactorrequirements. However, amino acid sequencing has uncovered extraordinary
variety among restriction enzymes and revealed that at the molecular level there
are many more than three different kinds. Type II enzymes cut DNA at defined
positions close to or within their recognition sequences. They produce discrete
restriction fragments and distinct gel banding patterns, and they are the only
class used in the laboratory for DNA analysis and gene cloning.
Unit Determination Assay
It is important to know the unit determination assay of the enzyme, one unit of
restriction endonuclease is defined as the amount of enzyme required to digest
one microgram of the appropriate substrate DNA completely in 60 minutes under
the conditions specified for that enzyme.
Practical Genetics 1st ed. 2008
33
Exercixe 7
Restriction Enzyme Digestion & Southern Blotting of DNA
Digestion by Restriction Enzyme
Materials
• DNA sample
• HinfI Restriction Enzyme
• Restriction enzyme buffer mix
• Nuclease-free water
Methods
Measure the DNA concentration
Use the Nano-drop spectrophotometer to measure the concentration of DNA, this
is used to determine the amount of HinfI restriction enzyme to be used.
Digestion of DNA
Mix the following components in a clean microtube
Component
nuclease-free water
10X Buffer R
DNA (0.5-1 µg/µl)
HinfI (10 u/µl)
Volume
16 µl
2 µl
1 µl
0.5-2 µl *
* The amount of the enzyme to be used depends on the concentration of DNA.
Mix gently and spin down for a few seconds.
Incubate at 37°C for 16 hours.
Analysis of DNA digestion
Analyze products on 2% agarose gel containing ethidium bromide.
Samples are prepared with loading dye and then loaded on the gel.
Visualize the PCR product on UV transilluminator.
Undigested DNA is represented by a sharp band near the wells of the gel, while
smearing indicates digested DNA sample.
Practical Genetics 1st ed. 2008
34
Exercixe 7
Restriction Enzyme Digestion & Southern Blotting of DNA
Southern Blotting
A technique used in molecular biology to check for the presence of a particular
DNA sequence in a DNA sample. The technique was developed by E.M.
Southern in 1975. Southern Blotting could be used to locate a particular gene
within an entire genome. The amount of DNA needed for this technique is
dependent on the size and specific activity of the probe.
The total cellular DNA of an organism (genome) or the cellular content of RNA
are complex mixtures of different nucleic acid sequences. Restriction digest of a
complex genome can generate millions of specific restriction fragments and there
can be several fragments of exactly the same size which will not be separated
from each other by electrophoresis. Southern Blotting techniques have been
devised to identify specific nucleic acids in these complex mixtures
The Southern Blot takes advantage of the fact that DNA fragments will stick to a
nylon or nitrocellulose membrane. DNA molecules are first elctrophoresed and
then transferred from an agarose gel onto a membrane The membrane is laid on
top of the agarose gel and absorbent material (e.g. paper towels or a sponge) is
placed on top. With time, the DNA fragments will travel from the gel to the
membrane by capillary action as surrounding liquid is drawn up to the absorbent
material on top. The membrane is now a mirror image of the agarose gel.
Procedure
Materials
• Whatman 3 mm Blotting Paper
• Nitrocellulose or nylon membrane filter
• Paper towels
• A weight
• 20x SSC (3M NaCl, 0.3M NaCitrate pH7.4)
• 2x SSC
• 6x SSC
• Labeled probe
Methods
Digest the DNA
DNA is digested as indicated before with Hinf1 restriction enzyme.
Electrophoresis
Load digested samples onto agarose gel (typically 0.8 to 1.0% agarose).
Run gel at maximum rate of 5 volts per cm.
Denature the DNA (usually while it is still on the gel).
Practical Genetics 1st ed. 2008
35
Exercixe 7
Restriction Enzyme Digestion & Southern Blotting of DNA
Soak it in about 0.5M NaOH, which would separate double-stranded DNA into
single-stranded DNA. Only ssDNA can transfer.
Transfer the denatured DNA to the membrane.
Traditionally, a nitrocellulose membrane is used, although nylon or a positively
charged nylon membrane may be used. Nitrocellulose typically has a binding
capacity of about 100µg/cm, while nylon has a binding capacity of about 500
µg/cm. Many scientists feel nylon is better since it binds more and is less fragile.
Transfer is usually done by capillary action, which takes several hours. Capillary
action transfer draws the buffer up by capillary action through the gel and into the
membrane, which will bind ssDNA. The Southern blot apparatus is illustrated in
Figure 7.2.
Note
Fill the container with 20x SSC so the level is just below the
You
may
use a vacuum
container edge.
blot
apparatus
instead
Place the gel onto the filter paper such that the open wells
of capillary action. In
are face down and the "back" of the gel is up. Make sure no
this
procedure,
a
bubbles are trapped between the gel and filters.
vacuum sucks SSC
Cut a piece of nitrocellulose to the exact size of the gel,
through the membrane.
This works similarly to
soak the filter in 6X SSC until it is fully wet.
capillary action, except
Place nitrocellulose on top of the gel - again take great care
more
SSC
goes
to insure that no bubbles are trapped between gel and
through the gel and
nitrocellulose.
membrane, so it is
Cut 4-6 pieces of Whatman 3 mm paper to the same size as
faster (about an hour).
gel and nitrocellulose.
Soak quickly in 6x SSC. Place these on top of nitrocellulose,
again watching for trapped bubbles.
Cut paper towels to same size as gel and filters - place these on top of the
stack.
Top with a weight.
Allow the blot to proceed, changing paper towels when the stack becomes
wet. Approximate blotting times is 14-16 hours (overnight ) for large DNA
Taking the blot apart
Peel off paper towels and Whatman 3 mm filters, trying not to dislodge the
nitrocellulose from the gel.
Flip the nitrocellulose/gel over and mark the positions of the gel wells onto
the nitrocellulose using a ball-point pen along the lower edge of the well.
After marking wells, peel the gel off and discard.
Rinse the filter in 6xSSC and air dry completely (~60 minutes at RT)
Blot Fixation
After you transfer your DNA to the membrane, treat it with UV light. This
cross links (via covalent bonds) the DNA to the membrane. (You can also
bake nitrocellulose at about 80oC for a couple of hours, but be aware that it
is very combustible.)
Practical Genetics 1st ed. 2008
36
Exercixe 7
Restriction Enzyme Digestion & Southern Blotting of DNA
Buffer
Figure 7.2. Setup of the Southern blotting apparatus.
Probe the membrane with labeled ssDNA (hybridization)
This process relies on the ssDNA hybridizing (annealing) to the DNA on the
membrane due to the binding of complementary strands. A probe is a fragment of
DNA of variable length (usually 100-1000 bases long), which is used to a
nucleotide sequences that are complementary to the sequence in the probe.
Must be labeled to be visualized, usually prepared by making a radioactive copy
of a DNA fragment. Probing is often done with 32P labeled ATP,
biotin/streptavidin or a bioluminescent probe.
To hybridize, use the same buffer as for prehybridization, but add your
specific probe.
Detection
Visualize your labeled target sequence. If radiolabeled 32P probe is used,
then you would visualize by autoradiography. Biotin/streptavidin detection is
done by colorimetric methods, and bioluminescent visualization uses
luminesence.
Practical Genetics 1st ed. 2008
37
8
Exercise 8: Plasmid DNA Isolation
Objectives
Extraction of plasmid DNA from E. Coli
Analysis of plasmid DNA by agarose gel electrophoresis and
spectrophotometer
Introduction
Many types of bacteria contain plasmid DNA. Plasmids are extrachromosomal, double-stranded circular DNA molecules generally containing
1,000 to 100,000 base pairs (Figure 8.1). Even the largest plasmids are
considerably smaller than the chromosomal DNA of the bacterium, which can
contain several million base pairs. Certain plasmids replicate independently of
the chromosomal DNA and can be present in hundreds of copies per cell. A
wide variety of genes have been discovered in plasmids. Some of them code
for antibiotic resistance and restriction enzymes. Plasmids are extremely
important tools in molecular cloning because they are useful in propagating
foreign genes. When plasmids are used for these purposes, they are referred
to as vectors.
Bacteria can sometimes take up DNA from the external environment, a natural
process that we call "transformation", and sometimes the DNA taken up is a
plasmid that can be maintained in the cell because it has an origin of
replication. Plasmids often make the bacteria gain a gene that gives a
selective advantage to the cell, which then replicates (instead of dying) and
makes more copies of the plasmid.
The plasmids used in transformation typically
have three important elements:
• A cloning site (a place to insert foreign DNAs)
• An origin of replication
• A selectable marker gene (e.g. resistance to
ampicillin)
Plasmids can be classified into 5 classes
according to their function:
1. Fertility-F-plasmids, Facilitate bacterial
conjugation.
2. Resistance-(R)plasmids, which contain
genes that can build a resistance against
antibiotics or poisons.
3. Col-plasmids, which contain genes that
code for bacteriocins, proteins that can kill
Figure 8.1. "A" represents a bacterial cell
containing plasmids. "B" represents electron
other bacteria.
micrograph of Plasmids.
4. Degradative plasmids, which enable the
digestion of unusual substances, e.g.,
toluene or salicylic acid.
5. Virulence plasmids, which turn the bacterium into a pathogen.
Practical Genetics 1st ed. 2008
Exercixe 8
Plasmid DNA Isolation
Isolation of plasmid DNA from bacterial cells is an essential step for many
molecular biology procedures. Many protocols for large- and small-scale
isolation of plasmids (mini-preps) have been published. The plasmid
purification procedures, unlike the procedures for purification of genomic DNA,
should involve removal not only of protein, but also another major impurity:
bacterial chromosomal DNA. To achieve separation of plasmid from
chromosomal DNA, these methods exploit the structural differences between
plasmid and chromosomal DNA. Plasmids are circular supercoiled DNA
molecules substantially smaller than bacterial chromosomal DNA.
Plasmid DNA isolation involves (1) growth of bacteria with amplification of
plasmid, (2) harvesting and lysis of bacteria, and (3) purification of plasmid
DNA. Normally there are ten to two hundred plasmids (relaxed - not
connected to chromosomal replication) per bacterial cell. The bacteria
containing the plasmid grow in LB medium plus antibiotic that selects for
plasmid (antibiotic resistant gene located on plasmid). By gentle bacterial lysis
small molecules, including covalently closed supercoiled plasmids are
released into solution. Mild alkali treatment to break most of the hydrogen
bonds in DNA and degrade chromosomal DNA. Closed circular plasmids
regain their native configuration when returned to neutral pH while larger
linear chromosomal DNA fragments remain in the denature state trapped in
the cell debris. The cell debris is precipitated and the supernatant containing
the plasmid is collected and plasmid is isolated. The yield of plasmid DNA is
dependent on the plasmid copy number, plasmid type, bacterial strain, and
growth conditions.
Procedure
Materials
LB (Luria-Bertani) containing 20 mg/l ampicilin
GTE buffer (50mM Glucose, 25 mM Tris-Cl & 10mM EDTA, pH 8)
NaOH/ SDS lysis solution (0.2 M NaOH, 1% SDS)
5 M potassium acetate solution (pH 4.8)
Isopropanol
TE buffer
RNAse solution (20 mg/ml stock)
PCIA (phenol/chloroform/isoamyl alcohol)
7.5 M ammonium acetate solution
Absolute ethanol
DNA Plasmid Miniprep Protocol
1.
Pick single colony and inoculate 5 ml of LB broth containing 20 mg/l
ampicillin or 0.1mg/5ml. Optional: Use a 15ml conical tube with a
loosened cap and a piece of tape to hold it in place. Shake at 250 RPM
37oC overnight.
2.
Centrifuge 1.5mL cells in 1.5 mL Eppendorf tube at top speed for 1
minute. Aspirate supernatant.
Practical Genetics 1st ed. 2008
39
Exercixe 8
Plasmid DNA Isolation
3.
Resuspend cell pellet in 100 ul of GTE buffer. Vortex gently if
necessary.
4.
Add 200 µl of NaOH/SDS lysis solution. Invert tube 6-8 times.
5.
IMMEDIATELY add 150 µl of 5 M potassium acetate solution (pH 4.8).
This solution neutralizes NaOH in the previous lysis step while
precipitating the genomic DNA and SDS in an insoluble white, rubbery
precipitate. Spin at top speed 1 min.
6.
Transfer supernatant to a new tube, being careful not to pick up any
white flakes. Precipitate the nucleic acids with 0.5mL of isopropanol on
ice for 10 minutes and centrifuge at top speed for 1 minute.
7.
Aspirate off all the isopropanol supernatant. Dissolve the pellet in 0.4
ml of TE buffer. Add 10µl of RNAse solution (20 mg/ml stock stored at 20 °C), vortex and incubate at 37°C for 20 to 30 minutes to digest
remaining RNA.
8.
Extract proteins from the plasmid DNA using PCIA (phenol/
chloroform/isoamyl alcohol) by adding about 0.3 ml. Vortex vigorously
for 30 seconds. Centrifuge at full speed for 5 minutes at room
temperature. Note organic PCIA layer will be at the bottom of the tube.
9.
Remove upper aqueous layer containing the plasmid DNA carefully
avoiding the white precipitated protein layer above the PCIA layer,
transferring to a clean 1.5 ml eppendorf tube.
10. Add 100 ml of 7.5 M ammonium acetate solution and 1 ml of absolute
ethanol to precipitate the plasmid DNA on ice for 10 minutes.
Centrifuge at full speed for 5 minutes at room temperature.
11. Aspirate off ethanol solution and resuspend or dissolve plasmid DNA
pellet in 50µl of DNA buffer.
12. Prepare a 1% agarose gel, load the plasmid DNA sample after mixing
with loading buffer and run the gel for 30 minutes.
13. Measure the concentration of the plasmid by a spectrophotometer.
14. The purity of the isolated plasmid DNA can also be measured by
spectrophotometer. DNA UV absorbance peaks at 260 nm, while
protein UV absorbance peaks at 280 nm. The ratio of the absorbance
at 260 nm/280 nm is a measure of the purity of a DNA sample from
protein contamination; it should be between 1.7 and 2.0.
The ratio of the absorbance at 260 nm/230 nm is a measure of the purity of
a DNA sample from organics and/or salts; it should be about 2.0. Low
260/230 ratio indicates contamination by organics and/or salts.
Practical Genetics 1st ed. 2008
40
9
Exercise 9: Transformation of Escherichia coli
Objectives:
Understand the transformation procedure using the heat shock method.
Understand how DNA can be transferred to an organism and the change
in phenotype that may result from such a transfer.
Transformation of a gene for resistance to the antibiotic ampicillin into a
bacterial strain (E. coli) that is sensitive to ampicillin.
Introduction
Transformation is a very basic technique that is used on a daily basis in a
molecular biological laboratory. The purpose of this technique is to introduce a
foreign plasmid into a bacteria and to use that bacteria to amplify the plasmid
in order to make large quantities of it. This is based on the natural function of
a plasmid, i.e. transfer genetic information vital to the survival of the bacteria.
In molecular biology, transformation refers to a form of genetic exchange in
which the genetic material carried by an individual cell is altered by
incorporation of foreign (exogenous) DNA. This foreign DNA may be derived
from unrelated species and even other kingdoms, such as bacteria, fungi,
plants or animals. Bacteria and yeast have been transformed with human
genes to produce proteins that are useful in treating human diseases and
disorders e.g. the production of insulin. Some bacteria have been modified
such that they are able to digest oil from accidental spills.
Transformation is usually more difficult with multicellular organisms such as
plants, in which it is necessary to either alter many cells with the new piece of
DNA or to alter just a single cell and then induce it to produce a whole, new
plant. Genetic transformation of plants and other organisms does occur
naturally.
The bacterium you will be transforming, Escherichia coli
(E.coli), Figure 9.1, lives in the human gut and is a
relatively simple and well understood organism. Its
genetic material consists mostly of one large circle of
DNA 3-5 million base pairs in length, with small loops of
DNA called plasmids, usually ranging from 5,000-10,000
base pairs in length, present in the cytoplasm. It is these
plasmids that bacteria can transfer back and forth,
allowing them to share genes among one another and
Figure 9.1. Electron micrograph
thus to naturally adapt to new environments. The ability
of a cluster of E. coli magnified
of bacteria to maintain these plasmids and replicate
10,000 times.
them during normal cell multiplication is the basis of cell
transformation. The plasmids are used as “gene taxis” in transformation
events to bring DNA of interest into the cell where it can integrate into the
genome (or remain as a plasmid within a bacterium) and be translated into
proteins not normally found in that organism.
The plasmid which is going to be used in this experiment contains an
ampicillin-resistance gene. Ampicillin is an antibiotic and works by preventing
Practical Genetics 1st ed. 2008
Exercixe 9
Transformation of Escherichia coli
E.coli from constructing cell walls, thereby killing the bacteria. When the
ampicillin-resistance gene is present it directs the production of an enzyme
that blocks the action of the ampicillin and the bacteria are able to survive.
Bacteria without the plasmid and hence the resistance gene are unable to
grow on a plate containing ampicillin in the medium and only the
transformants will survive.
Bacterial strains may have natural competence, i.e. they have the ability to
take up DNA from the medium. Natural competence is a genetically
programmed physiological state. Natural transformation is distinct from
artificial transformation by techniques such as electroporation. In addition,
some bacterial strains, such as E. coli, can be made artificially competent
using CaCl2 and heat shock treatment.
Since DNA is a very hydrophilic molecule, it won't normally pass through a
bacterial cell's membrane. In order to make bacteria take in the plasmid, they
must first be made "competent" to take up DNA. This is done by creating
small holes in the bacterial cells by suspending them in a solution with a high
concentration of calcium. DNA can then be forced into the cells by incubating
the cells and the DNA together on ice, placing them briefly at 42oC (heat
shock), and then putting them back on ice. This causes the bacteria to take in
the DNA. The cells are then plated out on antibiotic containing media.
The procedure to prepare competent cells can sometimes be tricky. Bacteria
aren't very stable when they have holes put in them, and they die easily. A
poorly performed procedure can result in cells that aren't very competent to
take up DNA. A well- performed procedure will result in very competent cells.
The competency of a stock of competent cells is determined by calculating
how many E. coli colonies are produced per microgram (10 -6 grams) of DNA
added. An excellent preparation of competent cells will give ~108 colonies per
µg. A poor preparation will be about 10 4 / µg or less. Our preps should be in
the range of 10 5 to 10 6.
Procedure
In this experiment you will be making competent cells, transforming them with
a plasmid and calculating their competency.
Materials
Luria-Bertani media
Luria-Bertani broth
50mM CaCl2 solution
Ampicillin (10 mg/ml)
Ampicillin resistant Plasmid (0.005 µg/µl)
Practical Genetics 1st ed. 2008
42
Exercixe 9
Transformation of Escherichia coli
Method
Competency
Label two sterile microtubes: one “+”and the other “-”
Using a disposable pipette, add 250 µl of 50mM CaCl2 solution to each
tube (“+” and “-”) and place them both on ice
Use a sterile plastic loop to transfer one or two 3 mm bacterial colonies
to the “+” tube, return tube to ice (Do not pick up any agar as it may
inhibit the transformation process)
Note
Transfer a mass of cells to the “-” tube
The cells are kept cold
Add 10 µl of ampicillin resistant plasmid directly into the to prevent them from
CaCl2 in “+” tube
growing
while
the
Return the “+” tube to the ice. DO NOT add the plasmid to plasmids are being
absorbed.
your "-" tube. Incubate both tubes on ice for 15 minutes
Plasmid Insertion
Remove the tubes from the ice and immediately hold them in a 42oC
water bath for 90 seconds. (The marked temperature change causes the
cells to readily absorb the plasmid DNA)
Move test tubes suddenly from the water bath back to the ice
Keep on ice for at least 1 minute
Recovery period
Allow the cell to regain strength and start to multiply
Add 250 µl sterile Luria Broth to both tubes using a sterile pipette
Move tubes to water bath at 37oC for 5 min
Growth & Isolation
Label four LB media plates as follows:
o Plate number 1, LB -no plasmid
o Plate number 2, LB/ Amp – no plasmid
o Plate number 3, LB + plasmid
o Plate number 4, LB/ Amp + no plasmid
Transfer 100 µl of cell suspension to each plate by using sterile transfer
pipette
Lift the lid of the plates and sweep the drop of cell suspension with a loop
to distribute it over the surface of the plate
Repeat this for the remaining 3 plates
Incubate the plates upside down for 24 hours at 37oC.
Analyze the results of the transformation
Determination of Transformation Efficiency
Transformation efficiency is a quantitative determination of how many cells
were transformed per 1 µg of plasmid DNA. In essence, it is an indicator of
how well the transformation experiment worked. You will calculate the
transformation efficiency from the data you collect from your experiment.
Practical Genetics 1st ed. 2008
43
Exercixe 9
Transformation of Escherichia coli
Count the number of colonies on the plate with ampicillin that is labeled
LB / Amp +
A convenient method to keep track of counted colonies is to mark the
colony with a lab marking pen on the outside of the plate
Determine the transformation efficiency using the formula:
Number of
transformants per µg
Practical Genetics 1st ed. 2008
=
final vol at recovery (ml)
vol plated (ml)
X
Number of transformants
µg of DNA
44
Exercise 10: SDS-PAGE
Objectives
To understand the principle of Sodium DodecylSulphate- PolyAcrylamide
Gel Electrophoresis (SDS-PAGE)
To become familiar with the SDS-PAGE setup
Introduction
A Polyacrylamide Gel is a separation matrix used in electrophoresis of
biomolecules, such as proteins or DNA fragments. Scientists used
polyacrylamide gels to separate DNA fragments differing by a single base-pair
in length. Most modern DNA separation methods now use agarose gels,
except for particularly small DNA fragments. It is currently most often used in
the field of immunology and protein analysis, often used to separate different
proteins or isomers of the same protein into separate bands. These can be
transferred onto a nitrocellulose to be probed with antibodies and
corresponding markers, such as in a western blot.
Proteins are a highly diversified class of biomolecules. Differences in their
chemical properties, such as charge, functional groups, shape, size and
solubility enable them to perform many biological functions. Determination of
the molecular weight of a protein is of fundamental importance to its
biochemical characterization. If the amino acid composition or sequence is
known, the exact molecular weight of a polypeptide can be calculated. SDS
gel electrophoresis is commonly used to obtain reliable molecular weight
estimates for denatured polypeptides.
A protein can have a net negative or net positive charge, depending on its
amino acid composition and the pH. At certain pH values of solutions, the
molecule can be electrically neutral, i.e. negative and positive charges are
balanced. In this case, the protein is isoelectric. In the presence of an
electrical field, proteins with net charges will migrate towards the electrodes of
opposite charge. Proteins exhibit different three-dimensional shapes and
folding patterns which are determined by their amino acid sequences and
intracellular processing.
The physical-chemical properties of proteins affect the way they migrate
during gel electrophoresis. Gels used in electrophoresis (e.g. agarose,
polyacrylamide) consist of microscopic pores of a defined size range that act
as a molecular sieve. Only molecules with net charge will migrate through the
gel when it is in an electric field. Small molecules pass through the pores
more easily than large ones. Molecules having more charge than others of the
same shape and size will migrate faster. Molecules of the same mass and
charge can have different shapes. In such cases, those with more compact
shape (sphere-like) will migrate through the gel more rapidly than those with
an elongated shape, like a rod. In summary, the charge, size and shape of a
native protein all affect its electrophoretic migration rates. Electrophoresis of
native proteins is useful in the clinical and immunological analysis of complex
Practical Genetics 1st ed. 2008
10
Exercixe 10
SDS-PAGE
biological samples, such as serum, but is not reliable to estimate molecular
weights.
Polyacrylamide gel electrophoresis
Polyacrylamide gels are formed by mixing the monomer,
acrylamide, the cross-linking agent, methylenebisacrylamide,
free radical generator, ammonium persulfate, and
tetramethylethylenediamine (TEMED) (TEMED accelerates
the rate of formation of free radicals from persulfate) in
aqueous buffer. Free radical polymerization of the acrylamide
occurs. At various points the acrylamide polymers are bridged
to each other (Figure 10.1). The pore size in polyacrylamide
gels is controlled by the gel concentration and the degree of
polymer cross-linking. The electrophoretic mobility of the
proteins is affected by the gel concentration. Higher
percentage gels are more suitable
for the separation of smaller
polypeptides. The polymerization
process is inhibited by oxygen.
Consequently, polyacr-ylamide gels
are most often prepared between
glass plates separated by strips
called spacers. As the liquid
acrylamide mixture is poured
between the plates, air is displaced
and polymerization proceeds more
rapidly.
Caution
It should be noted that
acrylamide is a neurontoxin and can be
absorbed through the
skin.
Sodium dodecylsulfate (SDS) binds
strongly to most proteins and
causes them to unfold to a random,
rod-like chains (Figure 10.2). No
covalent bonds are broken in this
process. The end result has two
Figure 10.1. Schematic diagram of polyacrylamide
important features:
polymer formation.
1) all proteins contain only primary
structure and 2) all proteins have a
large negative charge (Figure 10.2), which
means they will all migrate towards the positive
pole when placed in an electric field.
Proteins which contain several polypeptide
chains that are associated only by noncovalent
forces will be dissociated by SDS into separate,
denatured polypeptide chains. Proteins can
contain covalent crosslinks known as disulfide
bonds. High concentrations of reducing agents,
such as β-mercaptoethanol, can break disulfide
bonds.
Figure 10.2. A protein before adding SDS
and after adding SDS.
Practical Genetics 1st ed. 2008
46
Exercixe 10
SDS-PAGE
The amount of negative charge of the SDS is much more than the negative
and positive charges of the amino acid residues. The large quantity of bound
SDS efficiently masks the intrinsic changes in the protein. Consequently, SDS
denatured proteins are net negative and since the binding of the detergent is
proportional to the mass of the protein, the charge to mass ratio is constant.
The shape of SDS denatured proteins are all rod-like.
During SDS electrophoresis, the proteins migrate through the gel towards the
positive electrode at a rate that is inversely proportional to their molecular
weight. In other words, the smaller the denatured polypeptide, the faster it
migrates. The molecular weight of an unknown polypeptide is obtained by the
comparison of its position after electrophoresis to the positions of standard
SDS denatured proteins. The molecular weights of the standard proteins have
been previously determined. After proteins are visualized by staining and
destaining, their migration distance is measured. The log10 of the molecular
weights of the standard proteins are plotted versus their migration distance.
The molecular weight of unknowns are then easily calculated from the
standard curve.
Procedure
Materials
Acrylamide/ Bisacrylamide (40%)
Tris-HCl (1 M – pH 8.8)
Tris-HCl (0.5 M – pH 6.8)
Distilled water
10% SDS
10% Ammonium Persulfate
TEMED
Sample buffer
Silver stain kit
Methods
Label 2 beakers one "running" and the other "stacking"
The gel is prepared by adding the following components to the
corresponding beaker
Reagent
Acrylamide/ Bisacrylamide (40%) *
8% (Running Gel) 5% (Stacking Gel)
4.0 mls
2.5 mls
7.5 mls
7.5 mls
Tris-HCl
(1 M – pH 8.8)
(0.5 M – pH 6.8)
water (distilled)
8.2 mls
9.7 mls
10% SDS
200 µl
200 µl
10% Ammonium Persulfate
100 µl
100 µl
TEMED (added last)
10 µl
10 µl
* = 19:1 w:w ratio of acrylamide to N,N'-methylene bis-acrylamide
Mix ingredients gently in the order shown above, ensuring no air bubbles
form
Assemble two glass plates with two side spacers
Pour into glass plate assembly, first the running gel and then the
stacking gel
Practical Genetics 1st ed. 2008
47
Exercixe 10
SDS-PAGE
Insert comb, allow to set, and then remove comb after gel has set (15
min)
Assemble glass plates onto the electrophoresis unit, fill with
electrophoresis buffer
Dilute samples at least 1:4 with sample buffer, heat at 95oC for 4 minutes
prior to loading
Load the samples onto polyacrylamide gel
Run at 200 volts for 30-40 minutes
When the tracking dye reaches the bottom of the gel, turn off the power
supply
Pour out the buffer by inverting the entire unit over a sink
Open the gel sandwich. Remove the spacers and peel the gel off the
plate into a tray of stain. Wetting the gel helps to loosen it from the
plastic plate
Stain with Silver stain (follow the instructions of the kit)
Practical Genetics 1st ed. 2008
48