PCR

Question 1

Allele-Specific PCR

Answer 1

Used to detect SNPs
Uses SNP specific primers (matches only 1 allele)

Amplification if match, no product If mismatch

Question 2

Degenerate Primers and Primer Pools

Answer 2

Used for DNA barcoding when sequence is unknown

Question 3

Degenerate PCR

Answer 3

A mix of oligonucleotide sequences in which some positions contain a number of different possible bases, giving a population of primers with similar sequences that cover all possible nucleotide combinations for a given protein sequence

Question 4

Two-temp PCR

Answer 4

Low specificity annealing of primers initially

Useful for barcoding, pool of primers

Question 5

Touch-down PCR

Answer 5

Goal: increase specificity of primer binding

Good for genomic, good for areas where primers may tend to bind nonspecifically

Annealing temp gradually decreases until reach calculated Tm

Little product right away, but at high temp only a perfect match works at high temp

Question 6

Touch-up PCR

Answer 6

Goal: decrease specificity of primer

Tolerate initial mismatches

Question 7

Site Directed Mutagenesis (traditional)

Answer 7

Plasmid DNA

Design primers that contain mutation (substitutions, insertions, deletions)

Special taq: non-strand-displacing

Use DpnI - degrades methylated DNA (degrades parent DNA)

Plasmid is nicked, use special ultra competent E. coli

Question 8

Multi site directed mutagenesis

Answer 8

One reaction, one set of primers for each mutation

Digest with DPnI

Only substitutions

Question 9

Nested PCR

Answer 9

2 primer pairs

Increase likelihood of amplifying correct fragment (specificity)

Very low copy number DNA

Detect viral infection
Reduces false positives (whatever your amplifying is complimentary to four primers)

Studies of viral diversity, occult infection

Question 10

Inverse PCR

Answer 10

Used if you don’t know much about your goi sequence

Useful use retrovirus to insert dna into genome-where did it insert?

Cut up genome with RE (known site), self ligase into circles

Cut within known site, result = unknown DNA flanked by known sites (primers!)

Question 11

Reverse Transcription PCR (RT-PCR)

Answer 11

Isolation of rna from specific tissue

MRNA -> DNA via reverse transcriptase enzyme

Uses primer that binds to mRNA polyA tail

Traditional PCR used to amplify mRNA

OR all cDNA can be cloned into plasmids to make a cDNA library

Question 12

Real-time or Quantitative PCR (qPCR)

Answer 12

Detect product in real time

Proxy quantification method

1) dye incorporates into DNA, measured by machine (nonspecific, reads too high)
2) sequence-specific DNA probes (TaqMan and molecular beacons)

Question 13

TaqMan

Answer 13

Fluorophore, quencher in probe (complimentary, sits on gene) initially quenched

As taq comes along, cleaves/displaces fluorophore, fluorophore free to fluoresce

Amount of fluorescence relates to copy number

Expensive! Need a probe specific to every goi

Question 14

Molecular Beacon

Answer 14

Used in qPCR

Reporter is quenched when unbound

Probe binds to target, molecule “unfolds”, quencher too far away to block probe

Fluoresces when bound

Expensive, need unique probe

Question 15

Multiplex PCR

Answer 15

One tissue, want to know info about many genes

Primers for each gene

Add tissue, all sets of primers to rxn

Each set must have similar Tm, annealing temp, etc

Can add probes specific to each gene (each a different color) - tells you not only if something is present, but is a mutated form present? (Use colorimetric assay, can determine how much)

Can copy multiple organisms (which bacteria/pathogen is causing your symptoms)

Can be used to detect gene duplication

Question 16

Methylation PCR

Answer 16

Sodium Bisulfite Treatment

Methylation of promoter regions that turns those genes off

Heterochromatin = methylated = off

Treat sample, changes all C-> U

One set of primers recognizes methylated DNA, one set recognizes unmethylated