PCR, qRT-PCR, ISH Flashcards
Describe PCR and its purpose:
PCR amplifies DNA, enabling the generation of millions of copies of DNA in a very short time (DNA is very resistant to degradation).
Purpose is to make a huge number of copies of a gene.
Possible to analyse and characterise DNA fragments found in minute quantities.
Describe what PCR has been used to achieve:
Detect DNA sequence Diagnose genetic disease Carry out DNA fingerprinting Detect bacteria or viruses Research human evolution
Describe the two factors which made PCR a revolutionary technique:
Amplifies >1 billion copies of DNA from one template molecule
One day to genotype patient (much faster than Southern blot)
Describe how PCR can amplify small sections of DNA:
Makes many copies of small DNA fragment
Size range of DNA fragments amplified is 100-10,00bp (size allows for amplification of most genes)
Size and sequence of fragment defined by DNA primers (flank GoI)
>1x10^9 copies of small DNA fragments
Describe the two developments instrumental in maturation of PCR process:
- Heat-stable DNA taq polymerase
2. Thermal cycler development
Describe the analysis of PCR products (techniques):
By size fractionate DNA in agarose or polyacrylamide gels
Ethidium bromide stain DNA to determined the size of PCR product (dsDNA) and the amount of product (intensity of bands)
Use gel electrophosis
Describe multiplex PCR:
Amplified several DNA sequences at the same time
Uses several primer pairs:
1. All need the same melting temperature
2. All these primers should amplify DNA products that be a different size
Describe multiplex PCR:
Amplified several DNA sequences at the same time
Uses several primer pairs:
1. All need the same melting temperature
2. All these primers should amplify DNA products that be a different size
Describe reverse transcriptases and their two activities:
Encoded by retroviruses, copy viral RNA genome into DNA (convert mRNA to cDNA)
Has DNA polymerase activity and RNase-H activity (to hydrolyse template)
Taken from moloney murine leukemia virus or avian myeloblastosis virus
Which advantages of replication does RT-PCR take advantage of:
A DNA template
Nucleotides
Primers
Polyermase
Describe the templates for PCR:
Small amount of template
In theory a single molecules
Do not need to isolate sequence of interest
DNA template doesn’t need to be highly purified
DNA is stable in the absence of nucleases
Describe primers:
To use PCR, need to know sequences flanking GoI region
Primers provide specificity
Complementary to opposite strands with 3’ ends pointing towards each other
Should be in vast excess
Describe the importance of melting temperatures of primers:
Should have similar melting temperatures
Tm=2(A+T) + 4(G+C)
Tm= temperature at which half possible H bonds are formed (half primer bound to target)
Annealing temperature = Tm-5 degrees
List the PCR reagents:
Template (target) DNA 2 ss primers dNTPs PCR buffer (KCl, MgCl2, Tris pH 7-8) Thermostable DNA taq polymerase (KEY)
Name the advantages of PCR:
Sensitive: Exponential amplification from trace amounts of DNA
Fast: Can be done in a few hours
Safe: Non-radioactive method without toxic organic solvents
Name the disadvantages of PCr:
Contamination can be a serious problem, due to extreme sensitivity
Can sometimes be quite fickle and inconsistent
Name the problems with taq polymerase:
Does not have proof reading ability
Error rate 1 in 2x10^4 bases
Seems rare but recovered in cloning a single molecule
Newer polymerase (Pwo, Pfu) have high fidelity
List the future uses of PCR:
Molecular research and biotechnology:
Human genome project
Evolutionary studies
Analyse gene expression by measuring RNA levels (RT-PCR)
Detect presence of introduced gene (transgene)
List the future uses of PCR:
Molecular research and biotechnology:
Human genome project
Evolutionary studies
Analyse gene expression by measuring RNA levels (RT-PCR)
Detect presence of introduced gene (transgene)
Name ways that PCR can be used in medical diagnostics:
- Diagnosis and characterisation of infectious diseases (presence of viral pathogen, bacteria)
- Diagnosis and characterisation of human genetic diseases
- Diagnosis and characterisation of cancer cells
Name how PCR an be used in forensics:
Identify criminal suspects
Paternity cases
List some variations of basic PCR:
Allele-specific Assembly Asymmetric Digital Intersequence Inverse Ligation-mediated Nanoparticle assisted Miniprimer Nested Touchdown
