amplification

Question 1

what are the three distinct events during PCR cycles?

Answer 1

1. denature template
2. primers anneal
3. synthesis by thermostable polymerase

Question 2

why is the denaturation during the first cycle lengthened?

Answer 2

to make sure all dsDNA dissociates into single stranded

Question 3

what does heat change in PCR?

Answer 3

changes the conformation of polymerase to be active.

Question 4

what is the temperature and purpose of the denaturing step?

Answer 4

95-98 C, breaking apart dsDNA so primers can anneal

Question 5

why does the now ssDNA stay apart after cooling in the denaturing step?

Answer 5

because it would be a lot more energy to put them together than just binding to the random other stuff floating around

Question 6

T/F: primers must be PERFECTLY split to anneal to the template.

Answer 6

true

Question 7

when the reaction cools to 45-65 C what happens?

Answer 7

the primers hybridize to complementary single-stranded DNA.

Question 8

why do primers preferentially hybridize to complementary sequences.

Answer 8

primers are in much greater concentration than DNA and much shorter

Question 9

what is the last step of PCR?

Answer 9

DNA synthesis and DNA extension.

Question 10

how is DNA extension preformed?

Answer 10

extension of primer by thermostable polymerase

Question 11

how long is extension generally?

Answer 11

~1 min/kb

Question 12

what happens if DNA synthesis is too hot?

Answer 12

the proteins will be irreversibly damaged

Question 13

how many times is PCR repeated?

Answer 13

~30

Question 14

how many copies are generally produced during PCR?

Answer 14

10^12 usually

Question 15

what are the raw materials in PCR?

Answer 15

1. target template DNA
2. thermostable DNA polymerase
3. oligonucleotide primers
4. deoxynucleotide triphosphates
5. reaction buffer
6. magnesium ions
7. optional additives

Question 16

what two raw materials are paired together in PCR?

Answer 16

reaction buffers and magnesium ions

Question 17

generally how much template DNA is needed for a mammalian genome?

Answer 17

up to 5 ng

Question 18

T/F: for plasmid, mitochondrial, or chloroplast DNA you require less template DNA to get the same copies of template DNA.

Answer 18

true

Question 19

what kind of DNA polymerase is required for TaqPCR?

Answer 19

thermus aquaticus

Question 20

T/F: taq pol can incorporate errors.

Answer 20

true

Question 21

when is the incorporation of errors in taqPCR a problem?

Answer 21

when there is low template DNA causing errors in early cycles of PCR to get propogated

Question 22

what is the general primer length?

Answer 22

18-24

Question 23

what bases have a higher melting temperature?

Answer 23

G-C because 3 h bonds

Question 24

in an oligonucleotide primer how many G-C bonds should be in the sample

Answer 24

less than 50% of the primer

Question 25

what are the target sequences of oligonucleotide primers?

Answer 25

<2000 bp

Question 26

T/F: a primer should be extremely specific to one part of the genome

Answer 26

true

Question 27

T/F: to get a super specific primer it should then be extremely long to increase specificity.

Answer 27

false, too long a primer means the melting point might be close to or above 72C (aka extension temp of PCR)

Question 28

what can you do if there is too much primer?

Answer 28

slow down the reaction then test it on a gel seeing if you get one band

Question 29

how are oligonucleotide primers used?

Answer 29

in excess

Question 30

after 30 cycles of PCR most of the primers are where?

Answer 30

consumed by the reaction

Question 31

what are dNTPs?

Answer 31

raw materials for DNA amplification

Question 32

T/F: dNTPs are used in limited amounts to maximize PCR efficiency

Answer 32

false, they are used in non-limiting

Question 33

most applications use what concentation of each dNTP?

Answer 33

0.2 mM

Question 34

generating 10^12 copies of a 100 bp target sequence consumes how many dNTP molecules?

Answer 34

10^12*100 bp = 10^14 dNTP molecules

Question 35

what is Mg for in PCR?

Answer 35

affects the performance of Taq and the stringency of primer annealing

Question 36

Mg is the cofactor of what/in what?

Answer 36

enzymatic reaction of DNA polymerase

Question 37

in the absence of adequate free magnesium what happens?

Answer 37

taq pol is inactive

Question 38

in the excess of adequate free magnesium what happens?

Answer 38

increases level of nonspecific amplification

Question 39

what is an optional additive that binds potential PCR inhibitors?

Answer 39

Bovine Serum Albumin (BSA)

Question 40

a long initial denaturing step will do what?

Answer 40

dissociate high-molecular weight template strands

Question 41

the final extension step helps to add what onto the end of PCR products? why?

Answer 41

additional adenosine which makes scoring easier than if a product is a mixture of fragements with and without extra adenosine

Question 42

when should you keep going past 30 cycles of PCR?

Answer 42

in very low template DNA amounts available

Question 43

what happens when you keep going past 30 cycles in a normal sample?

Answer 43

often results in the amplification of undesired regions

Question 44

when should you stop PCR (on a graph)?

Answer 44

just before the plateau phase when you get a major and minor contributor

Question 45

T/F: you normally get a pure sample in PCR amp.

Answer 45

false

Question 46

what does the profiler plus cycling program do?

Answer 46

evens out locus specific variation.

Question 47

what does the touchdown cycling program do?

Answer 47

~50 cycles run at incrementally lower temperatures annealing above suspected melting temp gradually declining

Question 48

what does the touchdown cycling program ensure?

Answer 48

first primer-template hybridization events are stringent and keeping undesired amplification low.

Question 49

what is hot start PCR?

Answer 49

adding taq at higher temps

Question 50

T/F: taq pol is ALWAYS used.

Answer 50

false

Question 51

what kind of taq does most humans use?

Answer 51

proofreading taqs

Question 52

what 3 controls are needed in PCR?

Answer 52

1. negative 2. positive 3. primer

Question 53

what is a negative control?

Answer 53

there should be no DNA and no PCR products other than primer-dimers

Question 54

what is a positive control?

Answer 54

a sample known to work properly and amplifies well at the given locus

Question 55

what are the first controls to fail in PCR?

Answer 55

primer controls

Question 56

what does it mean when the controls look good and most samples look good but some didn't amplify?

Answer 56

some samples may have inhibitors or low templates

Question 57

what does it mean when no amplification of template control but the primer control reaction worked?

Answer 57

primer died halfway through

Question 58

what does it mean when neither the template control nor the primer control reaction worked?

Answer 58

you may have forgotten to add something important to your PCR

Question 59

what does it mean when there is an amplification of a negative control?

Answer 59

evalutate based on negative at beginning or end, reagents or sample may have been contaminated.

Question 60

what is a multiplex PCR?

Answer 60

amplifying many loci at once

Question 61

what is difficult to avoid in multiplex PCR?

Answer 61

primer-dimers

Question 62

what are the advantages and disadvantages of PCR?

Answer 62

- low amounts of DNA can provide product - does not have to be high molecular weight - contamination (can be an issue with low template samples) - PCR inhibitors - DNA mutations, primers will not anneal.

Question 63

what does whole genome amplification use?

Answer 63

polymerase and random hexamers as primers to amp the entire genome.

Question 64

what does the WGA enzyme do?

Answer 64

works by multiple displacement amplification or rolling circle amplification