RECOMBINANT DNA
Introduction
• Recombinant DNA
– A gene which has been constructed in the lab
using two pieces of DNA from different biological
sources
– The technology used to create and study the
molecules described
Cloning
• Insertion of a gene or other DNA sequence
into a bacterial cell, which will then be used
as a factory to produce millions of copies of
the gene of interest
Cloning
• Steps in cloning:
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Gene of interest is purified from cells or tissues
Restriction enzymes cut DNA
DNA fragments are ligated to a vector, such as a plasmid
Vector is transferred to a host cell
Host cell multiplies naturally into millions of cells, each
containing copies of the DNA
– Cloned DNA is purified from the host cells
OR…
– Gene product (protein) can be purified from host cells
Examples of Restriction Enzymes
Vectors
• Acceptors and carriers of DNA to be cloned
• Characteristics of vectors
– Contain several restriction sites
– Must be introduced into a host cell for replication of
vector
– Should have a selectable marker gene
– May contain nucleotide sequences that allow for
sequencing the inserted DNA
• Vectors are usually modified bacterial plasmids
Example of Plasmid
How do we know the cells took up the plasmid?
How do we know the plasmid contained the gene
of interest?
Example of Recombinant Plasmid
Replicating Inserted DNA
Clicker Question: How do you know
whether a host has taken up a vector
during a typical cloning experiment?
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A) It produces a blue colony
B) It produces a white colony
C) It produces any colony
D) It fails to grow at all
Clicker Question: What do you use to cut
a plasmid?
• A) An exonuclease
• B) A restriction endonuclease
• C) Tiny scissors
In Vitro DNA Amplification:
Polymerase Chain Reaction (PCR)
• PCR accelerated DNA duplication
compared with cloning
• Reaction components:
– Taq DNA polymerase
– Mg2+ ions
– Deoxyribonucleotide triphosphates (A,T,C,G)
– Primers specific for the DNA to be amplified
– Template DNA
– Optional: Reporter dye
In Vitro DNA Amplification: PCR
PCR Analysis
Reverse-Transcriptase (RT)-PCR
• Start with mRNA
– Isolate from tissue, cells, blood, plants,
bacteria, etc.
• Use reverse transcriptase to create cDNA
– RT: viral enzyme
– cDNA: complementary DNA
• Amplify cDNA using PCR
• Use to determine gene expression
After PCR…
(possibilities)
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Run the product (amplicon) on a gel
Compare CT values between groups
Purify the product
Sequence the product
Insert the product into a vector
Clicker Question: What is special about
Taq polymerase?
• A) It is especially fast at polymerizing
• B) It can proofread better than other DNA
polymerases
• C) It can bend the space-time continuum
• D) It can withstand high temperatures
Clicker Question: How does Dr. DuPriest
use PCR in her research?
• A) To create new vectors
• B) To determine size of fragments
• C) To compare gene expression between
groups
• D) To compare samples between a
suspect and a crime scene specimen
Restriction Mapping
Uses of Restriction Mapping
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Mapping a segment of DNA
Chromosomal analysis
Genetic testing (markers)
Forensic analysis – comparing DNA of
different individuals
Blotting
Types of Blotting
• Southern blotting
– Measures presence / amount of specific DNA
in a sample
• Northern blotting
– …specific RNA in a sample
• Western blotting
– …specific protein in a sample
• All use specific probes to detect specific
molecules (DNA/RNA/antibody)
DNA Sequencing
DNA Sequencing
DNA Sequencing
Clicker Question: Which type of
blotting has largely been
replaced by PCR?
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A) Southern
B) Northern
C) Western
D) Eastern
Clicker Question: DNA
sequencing has largely
replaced…
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A) Western blotting
B) Cloning
C) Restriction mapping
D) PCR