9) PCR Flashcards
What is PCR?
Polymerase Chain Reaction
It is a laboratory technique used to make multiple copies of a segment of DNA
Can start with template, and then will synthesise new identical copies
HAVE TO KNOW :The nucleotide sequences at the ends of a particular DNA region
What led to the development of PCR?
PCR: Developed by Kary Mullis in 1983
First use: Used to amplify the human B-globin gene
Originally used E.coli DNA but is not thermally stable= Denatured
Taq DNA polymerase specifically amplifies the target that is wanted= Its isolation from thermophilic bacterium was essential, as it is thermostable enzyme that was capable of repeat PCR cycling without need to add fresh DNA polymerase after each cycle
BUT: Taq polymerase tended to make mistakes
What are the 6 components needed for PCR?
1) DNA sequence of target region must be known
2) Template DNA for amplification
3) Primers
4) Thermostable DNA polymerase that is not inactivated by hearing to 95 degrees celsius
5) dNTPs: Deoxynucleoside triphosphates for each base (free nucleotides)
6) Thermal cycler= Machine that can be programmed to carry out heating and cooling
What is a primer and what are its key features?
Primers: Short oligonucleotides (short DNA or RNA molecules) that will bind to target sequences and prime DNA synthesis
Synthesis: ALWAYS from 3’ to 5’
Primer: should be 18-25 nucleotides in length
Similar melting temperature + GC content
GC clamp at the 3’ end to aid polymerisation initiation
Minimal secondary structure
Minimal repeats
Minimal runs of a single base (ideally no more than 4)
What are some potential primer problems?
1) Self complementarity or complementarity to other primer = overhangs that could prime DNA synthesis= hairpins, do not want primer dimers
What are the 3 steps of PCR? (NAMES only)
1) Denaturation
2) Annealing
3) Extension
What happens during 1) denaturation?
Denaturation: 90-95°C
High temperature= Separates the double-stranded DNA into single-stranded DNA
Result: Allows access for thermostable DNA polymerase and allows primers to bind
What happens during 2) Annealing?
Annealing= 55-65°C
1) Oligonucleotide primers can bind to regions next to the target gene to be amplified= 2 synthetic oligonucleotides complementary to the 3’ ends of the target DNA segment of interest are added
2) Added in great excess to denatured DNA= Preferentially anneal to the template rather than the complementary template strands simply reannealing to each other
3) Primers- Acts as starting point for the synthesis of new strands that will become copies of target region
What happens during 3) Extension?
Extension: 68-72°C
1) Optimum temperature for DNA polymerase= allow efficient incorporation of dNTPs and extend the primers into full length copies of target region
2) Polymerase will fall off when reach the end of template
How do you confirm that the PCR has applied a product of the correct size?
Sample can be loaded on an agarose gel
Size of PCR product= Estimated by comparing with a DNA molecular mass marker (ladder) which contains DNA molecules of known size and is loaded alongside the gel
DNA separated by electrophoresis= Negatively charged phosphodiester backbone of DNA= causes migration towards positive electrode
Larger DNA molecules move more slowly through the gel than smaller ones
What are some of the variations and applications of PCR in genetics, molecular biology and diagnostics?
1) Reverse transciptase PCR: Making complementary DNA copy of RNA by reverse transcription followed by PCR= can be used to determine quantities of mRNA or genome copies of RNA viruses
2) Allele-specific PCR: Primers are designed that will only amplify specific alleles of a gene
3) Methylation-specific PCR: Can distinguish between methylated and non-methylated DNA
4) Applications of PCR: Mutation screening, drug discovery, genomic cloning, genetic matching, DNA fingerprinting- tiny samples of DNA is isolated form a crime scene and can then be compared with DNA from suspects or with DNA database
5) Can be used for selective DNA isolation for cloning
6) Molecular archaeology: Ancient DNA can be isolated from archaeological specimens + DNA tends to degrade gradually
7) Molecular diagnostics: PCR can be used to identify any pathogen= specificity and rapid identification
