Define ATM
The temp at which 50% of the primer is bound
Principles of PCR
- denaturing
- anneal
- extension/ elongation
What happens when the temp is too low?
Non-specific primer binding
PCR reaction components
- DNA template
- DNA polymerase (Taq)
- forward primer
- reverse primer
- dNTPs
- buffer solution
- magnesium ions
- sterile distilled water
Stages of PCR reaction
- exponential
- leveling off
- plateau
Advantages of PCR
- analyses extremely small amounts of DNA
- amplify DNA for use in other applications
- modify DNA at specific locations
- determine sequence at unknown DNA fragments
Limitations of PCR
- contaminating DNA may produce spurious products
- intact template is essential
- product size limited by polymerase efficiency
- determining optimal parameters for combination of cycling and reaction mix can be tricky
Applications of PCR
- diagnosis f disease
- pathogen detection
- pre-natal screening
- genotyping
- genetic testing
- cancer screening
- cloning
Types of PCR
- nested PCR
- detection of methylated DNA
- reverse transcription PCR
- quantitative real time PCR
- multiplex PCR
PCR applications
- genotype/markers of inheritance
- identify abnormal copy number of gene
- point mutation detection
- allele specific PCR
- gene expression analysis
What does restriction fragment length polymorphism do?
Allows individuals to be identified based on unique patterns of restriction enzyme cutting sites in specific regions of DNA
How is RRLP done?
- amplification of a piece of DNA carrying a polymorphism
- cutting the product with restriction enzymes
- separating by agarose gel electrophoresis
- determining number of fragments and sizes
Uses of RFLP
- DNA fingerprinting
- markers of inheritance
How does haplotype analysis work?
Variable nucleotide tandem repeats are determined by PCR
Define multiplex PCR
Amplification of multiple targets in a single PCR experiment
Types of multiplex PCR
- single template
- multi template
Application of single template multiplex PCR
- detects deletions ins duchenne muscular dystrophy
- x and y deletions
Uses of multi template multiplex PCR
Detection of neurological and resp viruses
What is used to identify abnormal copy number of a gene?
Multiplex ligation-dependent probe amplification
- many probes, one template, one set of orimers
What is used for point mutations detection?
Sanger DNA sequencing
Process of sanger sequencing
- four different PCR reaction mixtures are prepared
- each has some ddNTP analogues to one of the four nucleotides
- DNA synthesis occurs until one of the analogues is incorporated,then it gets cut off
- many different length DNA strands
- primers labelled with 4 different coloured tags
- mixtures combined and added to single gel lane
- analysed using laser
How does allele specific PCR work?
- detects known SNPs
- primer only amplifies if 3’ end perfectly complements the variant
Definition of qRT PCR
Assay that monitors the accumulation of a DNA product from a PCR reaction in real time
Benefits of qRT PCR
- sensitive
- increased reproductability
- increased throughput
- cost efficient
- quantitative
