Recitation 3 (9/17)
Fly lab update - Score F2 adults this week in lab after finishing
your DNA fingerprinting PCR reactions. Careful not to dump
your media while anesthetizing. For questionable phenotypes,
show instructor or TA. When finished scoring anesthetized flies,
dump flies in morgue and clean out (with bottlebrush and water)
culture vials AND foam stoppers at the sink. Do not use soap!
Flush all culture media completely down drain. Remove and
dispose of labels. If you have questions, see your lab instructor
or me.
This week:
Human DNA Fingerprinting by PCR
Introduction to next week's lab:
Ø DNA fingerprinting = a method of using specific DNA primers that
amplify hypervariable minisatellite regions of the human genome.
These minisatellite loci are highly variable among humans such that if
several different minisatellites loci are analyzed, individuals will tend
to exhibit unique, multilocus genotypes or DNA "fingerprints."
Ø Minisatellite = STR (simple tandem repeats) = VNTR = variable
number tandem repeat locus = in the vertebrate genome, a class of loci
that are dispersed throughout the genome that consist of short repeated
copies of a DNA sequence that varies from 14 to 100 base nucleotides
long depending on the particular minisatellite locus. These repeat units
lie adjacent to one another (i.e., in tandem) on the chromosome and
alleles differ from one another by the number of repeat units at a locus
(between 14 and 40 repeats). (see figure below)
Ø Hyper variable = for a particular VNTR locus there are many alleles
(29 for the D1S80 locus we will be studying) in the human population
such that individuals often differ genotypically and therefore the locus
is said to be hypervariable.
Ø DNA primers = short (28-30 base) sequences of single stranded DNA
that are specific and complementary to the flanking regions around the
locus to be amplified (or replicated). The specificity of the primer
sequences is essential for the amplification of the appropriate gene.
DNA primers are produced synthetically by an oligonucleotide
synthesizer, which is a machine that can be programmed to make
DNA of a desired base sequence.
Ø Amplification = term referring to the process of replicating DNA
usually by the polymerase chain reaction (PCR).
Ø PCR = A relatively simple procedure that uses a machine known as a
thermal cycler and a special type of DNA polymerase called "Taq"
polymerase to amplify a particular DNA sequence. Chromosomal
DNA serves as the template for the replication and is combined with a
buffered solution of heat-stable Taq DNA polymerase, a specific pair
of oligonucleotide primers, the four deoxyribonucleotide building
blocks (dNTPs) of DNA, and the cofactor MgCl2. The PCR mixture is
placed in a DNA thermal cycler and programmed to undergo 30
cycles consisting of:
1 minute at 94oC, during which the chromosomal DNA is
denatured into single strands,
1 minute at 65oC, during which the primers hydrogen bond to
their complementary sequences on either side of the D1S80
locus, and
1 minute at 72oC, during which the Taq polymerase extends a
complementary DNA strand from each primer. (see figure
below and in lab manual)
Human DNA fingerprinting lab procedure
- Try to rinse food debris from your mouth before coming to lab
- In lab, each student obtain a conical tube with 10 ml saline label with your name
- Pour saline in your mouth and swish for 10 sec.
- Spit back into conical tube and spin
- Decant
- Resuspend in Chelex
- Boil
- Spin
- Pipet supernatant into clean, labeled tube
- Place on ice
- Prepare PCR reactions in tiny PCR tube - label tab on tube lid
with initials using fine-tip Sharpie - place on ice until all students
are ready
- The "PCR Mix" contains primers, dNTPs, Taq polymerase
buffer, and Taq polymerase
- You need to add PCR mix, MgCl2 and your DNA.
- Each lab will prepare a "negative control" reaction - What's
this?
- Instructor loads tubes into pre-programmed thermal cycler