PCR Flashcards
Learning Outcomes
After this lecture, students should be able to:
* list the components of a PCR and explain their roles in a PCR reaction.
* describe the steps in a PCR cycle and relate this to amplification of DNA.
* explain the binding of a PCR primer pair to a complementary double-stranded template and extension of the primers by DNA polymerases, including Taq DNA polymerase.
* describe cDNA synthesis, including the properties of the enzymes used.
* explain using examples the differences between PCR, RT-PCR and qRT-PCR and when each is used.
Obtaining DNA fragments
- DNA sequence is known, e.g. published in data bases, genome sequences
-buy the DNA fragments - All or part of the DNA sequence is known, only a related sequence is known
-Synthesise the DNA fragment using PCR - Uses DNA polymerases that can replicate DNA in vitro = in a test tube
DNA polymerases replicate DNA in vivo and in vitro
- DNA polymerase releases a pyrophosphate from dNTPs and adds the resulting dNMP to the 3’-OH end of the primer strand via a nucleophile attack mechanism
- Pyrophosphate is cleaved by pyrophosphatase → reaction is not reversible
- The new strand is synthesised in 5’ to 3’direction.
- The template strand is read in 3’ to 5 direction.
- In a PCR, we use a heat stable DNA Polymerase. Often Taq DNA polymerase from the thermophilic bacteria, e.g., thermus aguarcus (lives in not sorings) is used
DNA polymerases replicate DNA in vivo and in vitro photo
What do we need for a PCR?
- Template DNA - the double stranded DNA to be amplified
- Two primers - short, single stranded DNA oligonucleotides that can bind to either the upper or the lower template strand
- Taq DNA polymerase - the enzyme that synthesises copies of the template DNA
- dNTPs - dATP, dCP, dGTP and dTTP: building blocks for the newly synthesised DNAs
- Buffer - ensures that the reaction conditions are suitable for the amplitication
- Magnesium ions - required as cofactor for the Taq DNA polymerase. The enzyme cannot work with out it.
- A PCR machine - provides the correct temperatures and timing for individual PCR steps
Primers used for PCR
- Need sequence information to design primers
- Design primers so they can base pair with the template DNA strand
- 20-30 nucleotides in length
- Order those from companies that synthesize DNA primers
What do we need for a PCR? photo
Polymerase Chain reaction - many repeats (cycles) of three steps lead to large amplification of the template DNA photo
The power of PCR is in its duplication of DNA photo
Polymerase Chain reaction (PCR) - amplifying DNA in vitro
PCR takes place in a thermocycler
> PCR: in vitro method for generating large copy numbers of a specific
DNA fragment
invented by Kary Mullis in In 1983
None Prize in 144.31
using two primers, one to hybridize
in each cirand or a tarnet DNA
used DNA polymerase I fragment (Klenow fragment) from E. coli for exponential DNA replication. Today
we use thermostable DNA
0ovmerases.
Application of PCR: Cloning a DNA fragment photo
Applications of PCR
- Cloning of DNA, analysing DNA
- Genotyping and classification of organisms
- DNA fingerprinting - molecular marker of diseases, crime suspects, parental studies
- Mutational screening - identify mutations related to a trait, disease
- Site directed mutagenesis - create mutations and study the impact
- Detection of pathogens - identify a pathogen by a DNA marker
- Molecular epidemiology - follow a sequence indicative of a pathogen through a population
- Pre-natal diagnostics - identify risk factors
- Molecular archaeology - clone and sequence DNA from a fossil, relate to current species
- Molecular ecology - identify risk factors in the environment
- Gene expression studies
Application of Molecular PCR Markers in Forensic Science
- Parental studies
Molecular marker:
* Any site (locus) in the
genome of an organism at which the DNA base sequence varies among the different individuals of a population.
Application of Molecular PCR Markers in Forensic Science
- Parental studies
Use Reverse Transcription-Polymerase Chain Reaction (RT-PCR) to produce a DNA copy of an mRNA photo
Application of Molecular PCR Markers in Forensic Science
- Crime Scene Investigations
Reverse Transcription (RT) of RNA photo
Use Reverse Transcription-Polymerase Chain Reaction (RT-PCR) to produce a DNA copy of an mRNA
