Chapter 6

PCR and in vitro Mutagenesis

A. Basic features of PCR

1. PCR is a cell-free method of DNA cloning

• standard PCR reaction is a selective DNA amplification

• specificity of amplification and primer design

- nested PCR

- hot-start PCR

- touch-down PCR

2. Major advantages of PCR

• speed and ease of use

• sensitivity

• robustness

3. Major disadvantages of PCR

• flanking DNA sequences should be known

• short size and limiting amounts of PCR products

• Infidelity of DNA replication

4. Cell-based cloning of PCR products is needed for further characterization of the amplified DNA fragment

B. Applications of PCR:

1. PCR allows rapid amplification of a specific gene or exon of a gene as a first step for screening of uncharacterized mutations.

• PCR amplifications of specific exons from genomic DNA

• RT-PCR can be used for transcript analysis

2. PCR permits rapid genotyping for polymorphic markers

• use PCR as an alternative to RFLP by designing primers that flank the polymorphic restriction site. Following PCR amplification, the product is digested with the diagnostic restriction enzyme.

• Short tandem repeat polymorphisms (STRPs) known as microsatellite markers which are short sequences 1-4 nucleotides long tandemly repeated several times and often characterized by many alleles. Primers are designed from sequences flanking the repeats.

3. PCR can be used to assay for known mutations

a) allelic discrimination by size or susceptibility to restriction enzymes

- small deletions or insertions in alleles can be detected by PCR (e.d. deletions in the allele causing common cystic fibrosis (CFTR)

- a mutation that changes a restriction site can be detected as described before by PCR then digestion of the amplified fragment by the diagnostic restriction enzyme.

b) allelic discrimination by susceptibility to an artificially introduced restriction site

- One of the primer pair is designed to a mismatched nucleotide which together with the mutant nucleotide will create a restriction site. This restriction site will NOT be created in the wild type allele.

c) Allele-specific PCR (ARMS test)

- two primers can be designed, one specific for the mutant allele and the other specific for the wild type allele. The mutant specific primer ends with the mutant base at its 3’ end while the wild type specific allele ends with the normal base at its 3’ end. This method is used for detecting specific pathogenic mutations and was called amplification reffractory amplification system (ARMS).

d) mutation detection using the 5’ --> 3’ exonuclease activity of Taq DNA polymerase (TaqMan assay)

- A pair of primers P1 and P2 are used so that they are allele specific. The third primer P3 carries a fluorogenic group R and ends with a quencher group.

4. Degenerate PCR

- DOP-PCR permits DNA cloning using degenerate oligonucleotides.

- Linker-primed PCR (ligation adaptor PCR) permits indiscriminate amplification AND cloning of DNA sequences in a digested complex target DNA.

5. Anchored PCR is used to do ‘genomic walking’ upstream of a known sequence in the genome.

- This is a useful method to clone 5’ upstream regulatory sequences (promoter) of a gene whose full length cDNA sequence is known.