Strand A Flashcards
What is PCR?
It is the amplification of DNA
Based on DNA polymerase being able to synthesise a new strand
Starts with single stranded DNA
Taq polymerase used
Exponential increase in strands
What ingredients are used in eukaryotic replication?
Template DNA
40 or more proteins
Helicase
Primase
Polymerases
Nuclease
Ligase
ssDNA binding proteins (protects DNA)
Sliding clamps (polymerase doesnt fall off)
Why is PCR used?
Sensitive - amplify as little as one molecule of DNA
Specific - amplify a unique target sequence (temperature and Mg2+ specific)
Cheap
Rapid - few hours
Robust - DNA is very stable can be amplified from old degraded samples
What is typically in a PCR reaction tube?
Template dsDNA
2 primers to prime synthesis
Polymerase to copy the template
dNTPs
Magnesium as a co-factor
Buffer to maintain pH and provide necessary salt
How do nucleotides act as building blocks?
They contain functional groups that form bonds to create the copies
What is the function of taq polymerase?
Copy DNA accurately
Used for: synthesis, proof reading and primer removal
How are primers designed?
2 primers (forward and reverse)
Single stranded DNA
Length 18-24 (too long = hybridise too slowly, too short = not specific)
40-60% G/C content
Start and end with 1-2 G/C pairs
Melting temperate 50-60C
3’ end must be complementary to template DNA
Primer pairs should not have complementary regions
What is the function of magnesium in PCR?
It acts as a co-factor and a non-protein component that enables the activity of the catalysis
It enhances the enzymatic activity supporting DNA application
What is the function of a buffer in PCR?
Optimal pH is 8-9.5
Tris HCl
Potassium ions - promotes annealing
May be replaced by ammonium sulphate (destabilises base pairing bonds)
How many cycles are in PCR?
~30
What PCR products can be identified on a gel?
Molecular weight markers
PCR products
Primers
Template
What is an example of PCR in biotechnology?
Manipulate DNA - artificial fragments and then introduce into host cells and gets incorporated into genome
Knock out genes
Fuse host proteins with green fluorescent
How is reverse transcriptase PCR carried out?
Converts RNA to cDNA, use reverse transcriptase that converts RNA to DNA
Amplify DNA by PCR
What can be the source of the RNA for RT PCR?
Gene expression (mRNA)
RNA virus
What are the differences between end point PCR and real time PCR (qPCR)?
End point: Cheap
Semi-quantitive (band intensity)
Sequencing, genotyping and cloning
See results at the end
Real time: More expensive
Quantity PCR proportional to amount of template
Quantification gene expression, microarray verification, quality control, assay validation etc
Measures at exponential phase (more precise)
What are the different reagents used in qPCR?
SYBR green and TaqMan
How does SYBR green work?
Binds to the grooves in the DNA which increases the fluorescence
How does TaqMan work?
Probes are used with a fluorescence reporter and quencher
How does fluorescence quantify qPCR?
Using at threshold line at the cycle threshold (Ct)
A lower Ct = more cDNA in sample
Housekeeping gene
Standard curve
Standard points
What are housekeeping genes?
They are genes with a constant level of expression which are essential to support validity of qPCR
Confirms RNA extraction was good and efficient
Supports conclusions of expression levels
What is Nancy520 and how does it work?
Intercalating agent that goes between base pairs to allow the detection of PCR products
Why is PCR clinically valuable?
Sensitive
Specific (unique targets)
Relatively cheap
Rapid (only a few hours)
Robust - DNA is very stable
What are the different uses of PCR when determining diagnosis and prognosis?
Genotyping the patient - genetic traits, carriers, tissue matching and predicting response to drugs
Genotyping the pathogen - diagnosis of species and strain
Phenotyping the disease - measuring disease progression and disease severity
What is genotyping the patient?
Detecting which alleles an individual carries for a specific gene
What can sources of DNA be to genotype a patient?
Blood
Hair
Buccal smear
Cells from amniotic fluid
What are the 2 PCR based techniques for genotyping an individual?
PCR-RFLP (restriction fragment polymorphism)
ARMS-PCR (amplification refractory mutation system)
How does PCR-RFLP work?
Identifies allelic variants based on presence/absence of restriction site
What is the process of PCR-RFLP?
Allele 1 - healthy, Allele 2 - diseased
1 - no restriction site, 2 - has restriction site
Results during electrophoresis produce a heterozygous genotype
What are the advantages and disadvantages of PCR-RFLP?
Advantages: Cheap
Easy design
Applied to microindels and SNPs
Simple resources
Commonly used technique
Disadvantages:
Only possible with known RE site
RE expensive
Single nucleotide variation
Hands on and time consuming
Not suitable for high throughput
How does ARMS-PCR work?
Detects allelic variants using allele specific primers (only point mutations)
What is the process of ARMS-PCR?
2 Allele specific primers and conserved provers are used to amplify the DNA
Amplification takes place on which provides no amplification with one of the alleles but not the other
1 band will show up one will not
What are the differences between RFLP and ARMS?
RFLP: Uses locus specific primers
Relies on presence or absence of a restriction site to distinguish between variants
ARMS: Allele specific primers
Relies on stringency of PCR to distinguish
Alternative Tetra Primer ARMS-PCR, additional non-allele specific primers
What is genotyping the pathogen?
Identifying the species and strain of an infectious pathogen
Where can DNA/RNA be obtained to genotype a pathogen?
Blood
Sputum
Urine
Faeces
Skin swab
Tissue biopsy
What is the advantage of using PCR to diagnose microbes?
Vs microscopy: sensitive - detect single copies
Specific - identifies species and strain
Vs culture: sensitive - no need culture
PCR is quick
Vs patient antibody response: detects DNA/RNA therefore not dependent on immune response
How is a disease phenotypes?
To allow a snapshot of the disease status, severity or progression
What is a key technique to phenotype a disease?
Quantitive PCR - measuring the abundance of DNA/RNA in a clinical sample
What does ARMS-PCR stand for?
Amplification Refractory Mutation System
