Lecture 10 - PCR and sequencing Flashcards
Who determined the order of nucleotides in pieces of DNA?
Gilbert and Sanger
What does Sanger sequencing rely on?
The incorporation of dideoxynucleotides into newly replicated DNA
What is a dideoxynucleotide?
Terminator
Lacks a 3’ hydroxyl so is a terminator of chain extension
Allows no more nucleotides to be added
What is needed for Sanger’s dideoxy sequencing method?
Single stranded template DNA
Primer complementary to template
DNA polymerase
Pool of normal deoxynucleotides
Small proportion of radioactively-;abelled ddATP
What is the practice of the Sanger sequencing method?
- Add template + primer + DNA Pol I + dNTPs
- Add appropriate ddNTP
- Separate nested fragments on basis of size by electrophoresis
- Autoradiograph and read sequence from bottom upwards
- Looking for competition between normal nucleotides and incorporation of added product
What is the process of automates dideoxy sequencing?
- Template + primer + DNA Pol + all dNTPs + all fluourescent ddNTPs in one tube
- Dye present in each synthesized fragment corresponds to dye attached to dideoxynucleotide that terminated the synthesis of that particular fragment
- Pass nested product through an electrophoretic system and read with lasers
Who discovered the PCR?
Kary Mullis
What did Mullis say were the components of PCR?
Template dsDNA with target area
2 specific oligodeoxynucleotide primers
dNTPs
Buffer and MgCl2
Taq polymerase
Why is Taq polymerase useful?
Thermal stability of Taq DNA polymerase
What is the method of PCR?
- Heat to 94 degrees to denature DNA and cause H bonds to break
- Cool down (45-65 degrees) to allow primers to anneal
- Warm again to 72 degrees to allow primers to be extended
Repeat 30 times
What happens in cycle 1 of PCR?
- Target DNA is denatured
- Reaction is cooled, allowing primers to anneal to their complementary sequences
- Reaction is raised to 72 degrees, allowing Taq polymerase to extend the primers
- Products of cycle 1 can now act as substrates for cycle 2
How does PCR have both specificity and sensitivity?
Specificity provided by primers:
- Complementary to opposite strands with 3’ ends pointing towards each other
Sensitivity: one target molecule can be amplified to >10^9 molecules in just a few hours, product in one acts as template for next
Why do the regions defined by primers increase exponentially?
Starting DNA and hybrid duplexes also acts a templates for more PCR amplification
Expect >10^9 molecules of product from each starting target DNA after 30 cycles
What are the applications of PCR?
Medical-genetic purposes
Analysing pregnancy tissue for pre-natal genetic screening
Archaeological and ancient DNA sources
Use PCR to alter a DNA sequence
Forensic applications
How were the sequencing on archaic hominin genomes useful?
Found Europeans and Asians have 4-5% of their genes derived from Neanderthals
Gene flow can be detected from Neanderthals into modern humans
(later version: Europeans share 2% of DNA with Neanderthals)
