1: Fundamentals of PCR (MIDTERMS)

Question 1

The process of making/amplifying DNA copies outside of the body/ in vitro

Answer 1

Polymerase Chain Reaction (PCR)

Question 2

Vitro means?

Answer 2

Outside of the body

Question 3

How many kilobase of DNA are amplified in most PCR methods

Answer 3

up to 10 kbs

Question 4

PCR mirrors what mechanism in its procedure?

Answer 4

DNA replication

Question 5

What are the 2 reasons why we perform PCR?

Answer 5

1. DNA fingerprint (for suspect identification)
2. Disease identification (analysis of diseases)

Question 6

Enumerate the 3 main steps of PCR

Answer 6

1. Denaturing
2. Primer Annealing/Binding
3. Elongation/extension

Question 7

3 main step of PCR:

→ DNA is subjected at 95ºC for 30 to 60 seconds in order to separate/unwind

→ Mimics the function of helicase in cells

Answer 7

Denaturation of DNA

Question 8

What temperature and how many seconds is the process of denaturation of DNA in PCR?

Answer 8

95ºC for 30 to 60 seconds

Question 9

T or F

Denaturation of DNA is a step in PCR that can be skipped and is not important

Answer 9

F (CANT BE SKIPPED since it is important for DNA to be exposed and primers to bind)

Question 10

3 main step of PCR:

→ The primers bind to their complementary sequences on the single DNA strands

Answer 10

Primer annealing/binding

Question 11

T or F

In primer annealing/binding , primers bind to target sequence and are chosen based on similarand non complementary relationship with target sequence

Answer 11

F (primers are chosen based on their COMPLEMENTARY BINDING relationship with target sequence)

Question 12

What primers exist in PCR?

Answer 12

Forward and Reverse primers

Question 13

Temperatures for primer annealing depends on what?

Answer 13

G+C content (guanine and cytosine)

Question 14

T or F

Higher g+c content, higher melting temperature

Answer 14

T

Question 16

Indication of melting temperature of DNA?

Answer 16

If half of the strands are in single-stranded state

Question 17

3 main step of PCR:

→ at 72ºC, DNA polymerase adds nucleotides to the 3’ ends of the primers and is extended

Answer 17

Elongation/extension

Question 18

3 main step of PCR: Elongation

These are building blocks of DNA polymerase

Answer 18

dNTPS

Question 19

What temperature in elongation/extension process of DNA in PCR?

Answer 19

72ºC

Question 20

3 main step of PCR: Elongation

DNA polymerase adds nucleotides where?

Answer 20

3’ ends of the primers

Question 21

3 main step of PCR: Elongation

In what direction does DNA polymerase run along?

Answer 21

5’→3’ direction

Question 22

3 main step of PCR: Elongation

What strand direction favors the polymerase?

Answer 22

strand running on 3’→5’

Question 23

T or F

Extension starts at primers and attaches at appropriate nucleotide

Answer 23

T

Question 24

Final extension cycle runs for how long at what temperature

Answer 24

10 minutes at 72ºC followed by incubation at 4ºC

Question 25

T or F DNA polymerase typically functions at 80ºC

Answer 25

F (37ºC, anything lower or higher could cause it to not perform its function well)

Question 26

This polymerase used in PCR is a heat stable bacteria purified from hot springs in 1976

Answer 26

Thermus aquaticus (Taq DNA polymerase)

Question 27

T or F Size of the DNA fragment produced is dependent on size of the polymerase

Answer 27

F (dependent on the size of the PRIMER; longer sequence, longer primers)

Question 28

At each PCR cycle, the number of DNA molecules is increased to how many?

Answer 28

Doubled

Question 29

If you subject your target DNA to 10 cycles, you end up with how many copies of DNA?

Answer 29

1, 024 copies of DNA (2^n lng bb)

Question 30

If you subject your target DNA to 7 cycles, you end up with how many copies of DNA?

Answer 30

128 copies of DNA (2^n lng bb)

Question 31

T or F If you start with 2 unwound strands, first cycle produce 4 strands of DNA, While 3rd produces 8

Answer 31

T (basta doubled lang bb)

Question 32

Produced DNA depends on?

Answer 32

number of cycles set for thermal cycler

Question 33

Temperature and time for primers annealing/binding?

Answer 33

40-65 ºC, 30-60 seconds

Question 34

3 main step of PCR: Primers annealing/binding T or F temperature usually set for primers with higher g + c content are usually set 10 ºC-20 ºC below the melting temperature

Answer 34

T

Question 35

3 main step of PCR: Primers annealing/binding Higher g + c content = ?

Answer 35

Higher g + c content = higher melting temperature

Question 36

3 main step of PCR: Primers annealing/binding T or F the annealing temperature of DNA is where half of the strands are in a single-stranded state

Answer 36

F (the MELTING temperature of DNA is where half of the strands are in a single-stranded state

Question 37

thrives in hot temperatures hence it only needs to be added once at the beginning of the procedure

Answer 37

Thermus aquaticus Taq DNA polymerase)

Question 38

maximal enzymatic activity of Taq Polymerase?

Answer 38

75-80ºC

Question 39

DNA Polymerase functions at what temp?

Answer 39

37ºC

Question 40

Enumerate the process of PCR

Answer 40

1. Denaturation 2. Primer Annealing 3. Elongation

Question 41

functions ideally at 37ºC a. DNA Polymerase b. Taq Polymerase c. Both e. NOTA

Answer 41

a. DNA Polymerase

Question 42

95ºC for 30 to 60 seconds a. Annealing b. Elongation c. Both e. NOTA

Answer 42

e. NOTA (DENATURATION temp yan)

Question 43

40-65ºC for 30-60 seconds a. Annealing b. Elongation c. Both e. NOTA

Answer 43

a. Annealing

Question 44

72ºC for 10 mins a. Annealing b. Elongation c. Both e. NOTA

Answer 44

b. Elongation

Question 45

mimics function of helicase a. Elongation b. Denaturation c. Both e. NOTA

Answer 45

b. Denaturation

Question 46

requires melting temperature a. Annealing b. Elongation c. Both e. NOTA

Answer 46

a. Annealing

Question 47

95ºC for 5 mins (acc sa table) a. Initial Denaturation b. Denaturation c. Both d. NOTA

Answer 47

a. Initial Denaturation

Question 48

94ºC for 1 min (acc sa table) a. Initial Denaturation b. Denaturation c. Both d. NOTA

Answer 48

b. Denaturation

Question 49

55ºC for 1 min a. Annealing b. Denaturation c. Both d. NOTA

Answer 49

a. Annealing

Question 50

72ºC for 1 min a. Extension/Extension b. Final Elongation c. Both d. NOTA

Answer 50

c. Both

Question 51

4ºC to infinity a. Annealing b. Denaturation c. Both d. NOTA

Answer 51

d. NOTA (hold temp yn bb)

Question 52

What are the essential requirements for PCR?

Answer 52

1. Template dNA 2. Pair of Primers 3. Thermostable DNA polymerase 4. dNTPS 5. Cations and Buffer 6. Nuclease-free water 7. Thermocycler

Question 53

Range of length of primers?

Answer 53

15 to 30 nucleotides

Question 54

Essential requirements for PCR: → are synthetic oligonucleotides that serve to initiate DNA synthesis → short pieces of single-stranded DNA that are complementary to the 2 ends of each target DNA → each one provides the 3ʼOH group required to form a covalent bond (phosphodiester) with another nucleotide

Answer 54

Primers

Question 55

4 dNTPs used?

Answer 55

dTTP, dCTP, dGTP, and dATP

Question 56

recommended concntration of each dNTP?

Answer 56

200-250μM

Question 57

T or F Less than 4mM concentration of dNTP is inhibitory

Answer 57

F (MORE THAN 4mM concentration of dNTP is inhibitorykasi magcchelate mg2)

Question 58

are usually supplied with PCR enzymes at an amount that gives a working concentration of 1.5mM when diluted

Answer 58

Mg^2+

Question 59

may contaminate and degrade DNA including the primers, template, and amplified product

Answer 59

Nuclease

Question 60

T or F Nuclease-free water must be stored in small aliquots to reduce contamination

Answer 60

T

Question 61

a machine that automatically changes the temperatures and incubation times in each cycle

Answer 61

Thermocycler

Question 62

Optimal length of primer?

Answer 62

18-25 nucleotides

Question 63

15-30 nucleotides a. Range of primers b. Optimal length of primers c. NOTA

Answer 63

a. Range of primers

Question 64

18-25 nucleotides a. Range of primers b. Optimal length of primers c. NOTA

Answer 64

b. Optimal length of primers

Question 65

long repeats of any base are avoided to minimize?

Answer 65

secondary structures

Question 66

T or F minimize secondary structures by avoiding long repeats of any base as these cause a increase in available primers for cycling

Answer 66

F (minimize secondary structures by avoiding long repeats of any base as these cause a DECREASE in available primers for cycling)

Question 67

G+C content should be at what percent?

Answer 67

40-65%

Question 68

the final concentration of Mg^2 should be what amount?

Answer 68

0.5 to 5mM

Question 69

Higher concentration ofMg^2 = ?

Answer 69

Higher concentration ofMg^2 = higher product yield; lower specificity and fidelity

Question 70

Lower Mg^2 concentration =?

Answer 70

no PCR Product

Question 71

ideal concentration of dNTPs ?

Answer 71

200μM

Question 72

Higher dNTP = ?

Answer 72

Higher dNTP = chelates Mg^2--inhibiting DNA polymerase

Question 73

Lower dNTP = ?

Answer 73

HIGHER Fidelity; lowver/reduced yield