Molecular - PCR

Question 1

Process of PCR

Answer 1

Denature 95C, Primer binding 55, Taq polymerase at 72.

Question 2

Primer binding in several positions

Answer 2

Thermogradient cycler: 48 well plate, part is hot and part is cool; different annealing temperature. Avoid false priming and shows when it gets too hot: gives you ideal annealing temp

Question 3

MgCl

Answer 3

Stabilizes duplex (primer-template) as well as acting as cofactor. Also chelates nucleotides at high [ ]

Question 4

Trying to find

Answer 4

Optimal temp, magnesium concentration and nucleotide concentration

Question 5

Ways of calculating annealing temp

Answer 5

1. GC plus AT 4 + 2. For each G/C count 4C, each A/T count 2C. Works well for short oligos. 2. Tm a 64.9C + 41C x (number G and C in primer - 16.4)/N

Question 6

Why would 4 + 2 rule not work for certain lengths?

Answer 6

Too long so you get really big numbers. Temp would be so high that it would never anneal. This works best for 10-30bp

Question 7

Primer design

Answer 7

Want similar annealing temp for both primers; specificity; no hairpin loops; no homonucleotide runs especially at 3’ (might slip and therefore not get the right sequence); GC clamp

Question 8

GC clamp

Answer 8

G and C bind pretty well (3 H bonds each), which can aid primer binding at 3’ end

Question 9

Primer reverse compliment sequences

Answer 9

If you have a reverse compliment to the 3’ end of primer, it is possible that it will bind in the wrong direction and amplify the wrong stuff.

Question 10

Alternative to taq polymerase

Answer 10

Pfu: more accurate, used for PCR over 1000bp

Question 11

Multiplexing and microsatellites or STRs

Answer 11

usually 2-4bp that are multiplied. Represented as vertical lines —-|||||— ; target with primers in both directions; evolve like accordions. Powerful in population studies. Taq sometimes has problems with these too and some fainter bands can appear lower down.

Question 12

Microsatellite alleles

Answer 12

In this case an allele is how big a microsatellite is.

Question 13

Population study

Answer 13

Amplify a microsatellite locus in PCR for several individuals; run on gel. Difference in sizes are attributed to number of repeats. A person can be hetero- or homozygous.

Question 14

Use for microsatellites

Answer 14

Forensics (large number); population studies: help us determine amount of exchange b/w populations. Usually need a smaller number. Health: disease states, it’s not the locus but the allele which is correlated, helps zoom in.

Question 15

Sanger method

Answer 15

aka chain termination with fluorescent ddNTPs 1:100 dNTPs; electrophoresis; laser excitation and detection.

Question 16

Old school vs new school sequencing

Answer 16

Fluorescent dyes can all be done in one lane. Old school one lane had G terminator, one had C, etc

Question 17

AMI dye terminators

Answer 17

Each have fluorescein dye - the absorption is similar to the Argon laser; fluorescein basically absorbs the laser wavelength and donates this energy to individually modified dichloro-rhodamine (A, G, C, T)

Question 18

Limits

Answer 18

Big fragments (>1000bp) pass slowly, so peaks are wider and detector can count wrong; also it provides no info about enzyme rate potential

Question 19

Rate potential of enzyme - what is this??

Answer 19

How fast a polymerase enzyme is extending strand… Allow the enzyme to extend the strands more times each cycle (like for four minutes)

Question 20

How to detect microsatellites

Answer 20

Use a 4bp-cut site to digest genome; magnetic beads are attached to a sequence; fragments hybridize to beads; remove beads with magnet; heat up, removing fragments from beads; PCR; sequence fragments till you find one with repeats and sufficient space on each side for appropriate primers

Question 21

Detecting size of microsatellite

Answer 21

Use polyacrilimide. It can detect a difference of one repeat. Also need a marker so you can visulized it.