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Flashcards in Alternate PCR Deck (18)
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1

What can be changed in PCR?

Primers
Polymerase
Template
Target
Temperature cycle
Direction

2

What types of PCR change the template and temperature?

Colony PCR
Fast cycling PCR

3

Describe colony PCR

DNA isn't extracted from bacteria but the entire colony is added to the master mix
Performed using either a primer pair insert specific primers or plasmid specific primers that flank the insert
Used for rapid identification of correct ligation and insertion of inserted DNA in a plasmid

4

Describe fast-cycling PCR

Allows amplification of specific PCR products with significantly reduced cycling time
Buffer used increases the affinity of Taqp for short ssDNA thus reducing primer annealing time
2 step thermal profile- 94 denaturing and 68 annealing

5

What types of PCR change the polymerase?

High fidelity PCR
Hot-start PCR

6

Describe high fidelity PCR

Polymerase has a low error rate and significant binding affinity during amplification
Used in cloning, SNP analysis, and NGS applications

7

Describe hot-start PCR

Reduces occurrences of undesired products and formation of primer-dimers
Separate reagents until it reached denaturing temperature
Increased product yield
Takes less effort
Reduces risk of contamination

8

What types of PCR change the primers?

Multiplex PCR
Amplified fragment length polymorphism (AFLP) PCR
Intersequence specific PCR
Assembly PCR
Ligation mediated PCR
Asymmetric PCR
Inverse PCR
Miniprimer PCR
Nested PCR

9

Describe multiplex PCR

Multiple targets in one run
All primer pairs need similar annealing temperatures
Cheaper and quicker
Used in genotyping, mutation and polymorphism analysis, and pathogen detection

10

Describe AFLP PCR

Uses selective amplification of digested DNA fragments to generate unique fingerprints for genomes of interest
Generates large numbers of marker fragments without prior knowledge of the genomic sequence
Uses restriction enzymes and allows attachment of adaptors to sticky ends of fragments
Amplification using primers that are complementary to the adaptors
Then use agarose gel electrophoresis
Used to identify closely related species

11

Describe intersequence specific PCR

DNA fingerprinting that uses primers selected from specific segments repeated throughout a genome
Uses microsatellite sequences that are 16-25 bp long as primers in a single primer PCR reaction

12

Describe assembly PCR

Assembly of large DNA oligonucleotides from multiple shorter fragments
Used to make novel genes

13

Describe ligation-mediated PCR

Allows amplification without knowing the sequence of all the priming sites
Uses adaptors that are initially ligated to fragments of the target DNA
Primers the bind to the linker sequences

14

Describe asymmetric PCR

Used to preferentially amplify 1 strand of dsDNA
Achieved using an abundance of 1 primer

15

Describe inverse PCR

Used to amplify DNA when only limited sequence information is available
Restriction enzyme digestion, ligation
Results in a looped fragment which can act as a primer
Primers point away from each other

16

Describe miniprimer PCR

Uses engineered polymerase and 10 nucleotide miniprimers as short as 6 bp long
Used to amplify highly conserved DNA sequences like 16s or 18s rRNA

17

Describe nested PCR

The specificity of the reaction is enhanced by preventing the non-specific binding with the help of 2 sets of primers
The first set binds outside the target DNA and amplifies the larger fragment
The second set binds and amplifies only the target DNA
Highly sensitive to low concentrations of DNA

18

Describe qPCR

Gives DNA concentration
Real-time detection during the exponential phase
Uses fluorescent dye
Can be used with RT to determine expression level of genes