Please enable JavaScript.
Coggle requires JavaScript to display documents.
MM11 - Disease diagnosis molecular biology methods 1 (ii) (Polymerase…
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
e.g. BRAF: 1bp change @ codon 600, no DNA gained/lost, 50% melanoma patients have this (suitable for argeted therapy)
micro satellites
insertions/deletions
e.g. F508del - 3bp deletion, phenylalanine deleted, cf