pBR322 PCR

BACKGROUND

The polymerase chain reaction (PCR) was first developed in 1985. Quickly, it became one of the most used and useful tools available to molecular geneticists. As we have and will discuss in class, PCR has been used for a host of purposes. The importance of this technique to biomedical research was illustrated when Kary Mullis, the inventor of PCR, received the Nobel Prize in Chemistry in 1993

Basically, the polymerase chain reaction is an in vitro enzymatic technique which results in the exponential amplification of a targeted DNA sequence. The process consists of three main steps: denaturation, annealing, and extension.

Denaturation — double stranded DNA must be denatured so that each strand can serve as a template for the production of a complementary strand. Denaturation typically is achieved by heating DNA to 95^oC.

Annealing — entails addition of two specific oligonucleotide primers and their subsequent hybridization to target sequences within the denatured DNA. Annealing temperature depends on sequence and length of primers.

Extension — DNA synthesis. DNA polymerase adds new nucleotides to free 3’ ends of annealed primers, Temperature must be compatible with activity of enzyme.

For a primer on PCR, click here.

Oligonucleotide Primers

Generally, primers are 18-24 bases in length.
Approximately 50% GC content.
Both primers used should have similar Tms.
Annealing temperature generally 5^oC below Tm.
Primer should contain little internal homology.
Primers should contain a 3’ G or C
Generation of degenerate primers.
Degeneracy of genetic code
Codon usage tables for different species

Thermostable DNA polymerases

Taq — originally isolated from Thermus aquaticus.
Possesses 5’-3’ DNA synthesis activity.
Lacks 3’-5’ exonuclease (proofreading) activity.
VENT — isolated from Thermococcus litoralis.
Possesses proofreading activity.
Pfu — originally isolated from Pyrococcus furiosus.
Possesses proofreading activity.

Variations on a theme

Basic PCR — amplification of DNA using two primers complementary to ends of region to be amplified
RT-PCR — combined use of reverse transcription and PCR to amplify mRNA
PCR Cloning — primers designed to incorporate restriction enzyme sequences at their 5’ ends to facilitate cloning
PCR-based Site-directed mutagenesis — primers designed to incorporate a specific nucleotide mutation, thereby producing a mutated PCR product

GOALS

Amplify tetracycline resistance gene from pBR322 plasmid
Confirm identity of PCR product by gel electrophoresis and Southern blot
Ligate PCR product into another plasmid
Transform tetracycline sensitive E. coli with the recombinant plasmid
Confirm phenotype of transformed bacteria

MATERIALS

Template DNA: pBR322

Primer 1: 5’-CGCAGTCAGGCACCGTGTATG-3’
Primer 2: 5’-CCATTCAGGTCGAGGTGGCCC-3’

To a 0.5mL microfuge tube, add the following reagents:

NOTE: The PCR mix contains Taq DNA polymerase, dATP, dTTP, dCTP, dGTP, and a buffer.

Briefly centrifuge the tube to collect all material at the bottom.

Cycle as follows:

94^oC 2 minutes — 1 cycle

94oC 1 minute
50^oC 1 minute
72^oC 1 minute

30 cycles

Store amplified DNA at —20^oC.