MM11 - Disease diagnosis molecular biology methods 1 (ii)

Polymerase Chain Reaction (PCR)

amplification method - testing DNA requires a large amount, + PCR can produce millions of target copies

needed...

thermostable DNAP

can't be human as will denature when heated

e.g. thermus aquaticus DNAP

DNA template

nucleotides

Mg2+

2 oligonucleotide primers

1 per DNA strand

1 marks end, 1 marks beginning

can be designed thanks to human genome project

synthetic

18-20 bps

complement target dsDNA

25-35 cycles of 3 steps...

each cycle doubles no. of copies

exponential amplification

1) denature DNA @ 95 degrees

2) anneal primers (alter their properties with heat) @ 55 degrees - results in hybridisation with template)

3) replicate @ 72 degrees (DNA synthesis)

Sanger DNA sequencing

determines DNA sequence (base reading)

controlled PCR interruption

standard PCR with terminator dideoxyrobonucleotides (ddNTPs)

4 types for ATC and G

present in lower conc (1/100) than dNTPs

compete with dNTPs for hybridisation

fluorescently labelled (1 colour per type)

when ddNTPs win phosphodiester bind can't form (interruption)

used to detect small scale variations

SNPs

micro satellites

insertions/deletions

e.g. F508del - 3bp deletion, phenylalanine deleted, cf

e.g. BRAF: 1bp change @ codon 600, no DNA gained/lost, 50% melanoma patients have this (suitable for argeted therapy)