PCR

Question 1

PCR principle

Answer 1

selective amplification of a chosen region of a DNA molecule

Question 2

Known sequences in PCR

Answer 2

those at the borders of the region

Question 3

Short primers annealing to the template delimit

Answer 3

the region to be amplified

Question 4

Amplification carried out by

Answer 4

DNA polymerase

Question 5

PCR amplification is

Answer 5

exponential and non-stop

Question 6

Nf

Answer 6

N0(1+Y)^n

Question 7

n

Answer 7

of cycles

Question 8

N0

Answer 8

initial copy # of target sequence

Question 9

Nf

Answer 9

final copy # of amplified sequence

Question 10

Y

Answer 10

efficiency of amplification per cycle

Question 11

Steps in PCR cycle

Answer 11

1. Denaturation of dsDNA template,
2. annealing of primers
3. Extention: synthesis of complimentary DNA by DNA polymerase

Question 12

Sample PCR program

Answer 12

1. denaturations 95˚, 30 sec
2. denaturation 95˚ 30 sec
3. annealing 55˚ 60 sec
4. extension 72˚, 2.5 min
5. Repeat steps 2-4 30 times
6. Complete extension 72˚, 5 min

Question 13

Problems in chaining

Solutions to the problems

Answer 13

Inactivation of DNA polymerase I during the denaturation step, Different temperatures required for three steps,
Thermostable DNA polymerases able to withstand multiple cycles of denaturation, Programmable thermocyclers that provide precise temperatures

Question 14

Components of PCR

Answer 14

DNA template,
primer,
Taq polymerase and dNTPs

Question 15

DNA template requirements

Answer 15

Contains at least one copy of target sequence
1 pg of plasmid DNA to 1 µg of mammalian DNA
ss- or ds- DNA
Linear or circular DNA
free of contaminants that may inhibit the reaction

Question 16

Primer requirements

Answer 16

Quality (Automated DNA synthesizers, chromatographic or gel purification for more demanding applications)

Question 17

Primer design is one of

Answer 17

the most important factors for successful pcr

Question 18

Important factors in primer design:

Answer 18

length of the primer
having no significant homology with other sites on either strands of target DNA
not having self-complimentary sequences(within a primer or between primer pairs)

Question 19

Primer Lengths Generally between

Answer 19

17 and 25

Question 20

Shorter primers have increased likelyhood of

Answer 20

binding to non-targeted sites

Question 21

Longer primers have higher ________ but lower ________

Answer 21

specificity, annealing rate

Question 22

When annealing temperature is too high

Answer 22

primers and templates remain dissociated

Question 23

When annealing temperature is too low

Answer 23

There are mismatched hybrids and not all of the correct bp form

Question 24

With correct annealing temperatures

Answer 24

Priming occurs only at the desired target sites

Question 25

Tm

Answer 25

4 * (G+C) + 2*(A+T)

Question 26

Tm's for two primers need to be similar in order to

Answer 26

minimize the potential for mispriming

Question 27

optimal base compositions

Answer 27

~40-60% [G+C] content

Question 28

Annealing temp typically

Answer 28

~2-5 ˚C below Tm

Question 29

Annealing time depends on __________ but a ____________

Answer 29

length and complexity of the primers, fixed time is usually used

Question 30

Thermostable DNA polymerases are

Answer 30

isolated from thermophilic and hyperthermophilic archaebacteria, stable at high temperature, have a tradeoff between extension speed and proof-reading function

Question 31

Taq DNA polymerases

Answer 31

have faster extension are more tolerant of inhibitory substances tend to add a overhanging adenosine

Question 32

Pfu polymerases

Answer 32

lower error rate due to 3'-5' exonuclease activity can amplify longer fragments stronger thermal stability

Question 33

Ways to visualize and Identify PCR products

Answer 33

``` by size restriction analysis (Sites present in amplified DNA, Mutations) by sequencing (purification, template for sequencing) ```

Question 34

In T-A cloning, taq polymerase tends to

Answer 34

add a non-templated A to the 3' end

Question 35

In T-A cloning, cloning is into a special vector with

Answer 35

"T" overhangs(TA vector) for base-pairing with the ends of the pcr product

Question 36

polynucleotides synthesized from taq polymerase often have an

Answer 36

extra adenosine at their 3' end

Question 37

In preparing a TA vector Treat blunt cut vector with ______ Engineer a vector containing _____

Answer 37

Taq in the presence of dTTP | an Xcml site

Question 38

In cloning by restriction sites use _____________ in the DNA target introduce __________ at the _______ of the PCR primers Newly created sites must be _______________

Answer 38

naturally occurring restriction sites new restriction sites at the 5' end of the PCR primers absent from the amplified DNA fragment

Question 39

``` Allelle specific pcr There is at least ________ Design a primer having __________________ the mismatch is at ____________ Use a polymerase w/out _________ ```

Answer 39

one nucleotide difference between the two templates a perfect match with one allele, but a mismatch with the other allele the last nucleotide 3' -> 5' exonuclease activity

Question 40

Taq can only amplify fragments up to

Answer 40

3-5 kb

Question 41

Long PCR Combining two DNA polymerases allows Has an _____ but _______ "workhorse to _________ has small amounts of ________ with ___________ which removes ________

Answer 41

amplifation of DNA fragments up to 20-30kb efficient, error-prone, carry out most of the DNA synthesis proof-reading polymerase, 3'-5' exonuclease activity , mismatched bp

Question 42

Assembly PCR

Answer 42

synthesize longer DNA from two paritally overlapping primers or DNA sequences

Question 43

Gibson assembly

Answer 43

synthesize longer DNA from two or more overlapping primers or DNA fragments, cloning without restriction sites

Question 44

RT-PCR

Answer 44

determine the amount of RNA transcripts using cDNA as the template

Question 45

multiplex PCR

Answer 45

Apply two or more targets in the same reaction

Question 46

BRIDGE PCR DNA fragments are ____________ Has a solid surface coated with __________ First round of synthesis results in ________ With nucleotides and polymerase added ____ The end result is

Answer 46

- ligated w/ 2 adaptors - 2 types of primers corresponding to the adaptors - newly synthesized DNA having the 5' end bound to the solid surface - further amplification is realized only when anchored DNA forms a 'bridge' with a primer on the surface - localized amplification of a single molecule of DNA template

Question 47

Emulsion PCR, prepare a ________ by preparing an ________ containing _________ and adding the _______ to ________ while _________

Answer 47

water/oil emulsion - preparing an oil phase (w/ surfactant) - preparing an aqueous phase containing pcr products - aqueous solution to oil phase while stirring is taking place