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POLYMERASE CHAIN REACTION(PCR)

POLYMERASE CHAIN REACTION(PCR)

Introductory notes

What is a gene? The old concept:Gene is the inherited determinants of the phenotype which occupies a specific chromosomal locus.

What is a gene? The new concept:Gene is a DNA sequence that codes for a polypeptide.

Chromosome

Determinant

5’…TCGATCCTGTATTAACGAACTGACAATCCAGACT…3’3’…AGCTAGGACATAATTGCTTGACTGTTAGGTCTGA…5’

POLYMERASE CHAIN REACTION(PCR)

Objectives:a) to learn the technique of the PCR b) to amplify a small fragment from the Arabidopsis

thaliana TTG gene in order to clone it into a cloningvector

Arabidopsis thaliana (the thale cress) has five pairs of chromosomes (2n=10) with around 27,655 protein coding genes. The TTG gene is located in chromosome 5, about 29.5 cM from the tip of the chromosome.

ttg = transparent testa glabra1. No trichomes on leaves2. Lack of anthocyanin in leaf and stem epidermal cells (no

purple color when the plant is under a stress)3. Lack of mucilage and tannins in the seed coat

(seeds appear yellow, not brown)

The phenotypes associated with the ttg gene do not segregateindependently:

-controlled by a single gene-transcribed by a single mRNA -ttg is a pleiotropic gene

EMS-induced recessive mutation:results in CT transition; induces a terminationcodon (TAA) in the place of the codon 317 for the amino acid glutamine (CAA)

Size of the locus: 5777 bp (5.7 kb)Size of the gene: 2261 bp (3267-5527)Fragment to be cloned: 1299 bp (1.3 kb), located between

nucleotide pairs 2573 and 3872

The 1299 bp (1.3 kb) is picked from the wild-type strain of A.thaliana called Columbia.

5’

3’

3’

5’

TTG locusTTG gene

3267 5527

38722573

Region to be cloned(target)1299 bp

Begins

Ends

PCR amplificationPCR mimics DNA replication in the cell.

PCR (invented by Kary Mullis, Nobel Prize winner, 1993) allowsdirect amplification of a specific target DNA sequence within apopulation of DNA molecules.

In order to find the target region and amplify it, you need: a) information about the nucleotide sequence flanking the

target regionb) primers: single-stranded DNA complementary to the 3’

end of the target regionc) nucleotide mix (dNTP): contains A G T Cd) MgCl2, to assist annealing the primers; to create

working environment for Taq DNA polymerasee) Taq DNA polymerase (extracted from Thermus

aquaticus), for polymerization of nucleotides

PCR amplification mimics DNA replication in vivo.

PCR amplification mimics DNA replication in vivo.

5’

3’

3’

5’

Target region

Enzyme activation and DNA denaturation at 92°C

Annealing at 64°C or lower

RF

3’

3’

3’

3’

Extension at 72°C

R

F

RF

Product of the first cycle

3’

3’

3’

3’ 3’

3’

dNTP and polymerization by TaqG A T C C T A A A G

T C C G A T G C T A

___________________________________________________________________Block temp.

Step No. in °C Time Operation___________________________________________________________________

1 92 2 min Enzyme activating/denaturing2 92 30 s Subsequent denaturation3 47.5 1 min Annealing4 72 3 min Extending5 39 times to step 2 Cycle repeating6 4 0 h, 0 min, 0 s Holding at 4°C indefinitely7 End End of the program

__________________________________________________________________

Procedure

1. Program the thermal cycler.

2. Prepare the reaction.

-mix well-centrifuge briefly-transfer contents to a PCR tube; no bubbles!

3. Place the PCR tube in a thermal cycler.

DNA

ttg-F

dH2O

Taq

ttg-R

dNTP

Buffer

MgCl2