PCR Flashcards
0
Q
Define ATM
A
The temp at which 50% of the primer is bound
1
Q
Principles of PCR
A
- denaturing
- anneal
- extension/ elongation
2
Q
What happens when the temp is too low?
A
Non-specific primer binding
3
Q
PCR reaction components
A
- DNA template
- DNA polymerase (Taq)
- forward primer
- reverse primer
- dNTPs
- buffer solution
- magnesium ions
- sterile distilled water
4
Q
Stages of PCR reaction
A
- exponential
- leveling off
- plateau
5
Q
Advantages of PCR
A
- analyses extremely small amounts of DNA
- amplify DNA for use in other applications
- modify DNA at specific locations
- determine sequence at unknown DNA fragments
6
Q
Limitations of PCR
A
- 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
7
Q
Applications of PCR
A
- diagnosis f disease
- pathogen detection
- pre-natal screening
- genotyping
- genetic testing
- cancer screening
- cloning
8
Q
Types of PCR
A
- nested PCR
- detection of methylated DNA
- reverse transcription PCR
- quantitative real time PCR
- multiplex PCR
9
Q
PCR applications
A
- genotype/markers of inheritance
- identify abnormal copy number of gene
- point mutation detection
- allele specific PCR
- gene expression analysis
10
Q
What does restriction fragment length polymorphism do?
A
Allows individuals to be identified based on unique patterns of restriction enzyme cutting sites in specific regions of DNA
11
Q
How is RRLP done?
A
- 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
12
Q
Uses of RFLP
A
- DNA fingerprinting
- markers of inheritance
13
Q
How does haplotype analysis work?
A
Variable nucleotide tandem repeats are determined by PCR
14
Q
Define multiplex PCR
A
Amplification of multiple targets in a single PCR experiment
