PCR, Cloning, Plasmids (#3)

Question 1

synthesizes large quantities of DNA fragments

Answer 1

PCR

Question 2

PCR =

Answer 2

polymerase chain reaction

Question 3

application of PCR (4):

Answer 3

• species identification
• genotyping
• gene cloning
• forensics

Question 4

who came up with PCR and when?

Answer 4

Karry Mullis in 1983

Question 5

PCR reaction mix (5):

Answer 5

• target DNA
• primers
• thermostable DNA
• polyermase (Taq polymerase)
• deoxyribonucleotide triphosphates (dNTPs)

Question 6

part of PCR reaction mix: need at least ONE piece of it

Answer 6

target DNA

Question 7

short pieces of DNA made by genetic engineer; binds to complementary DNA after it’s denatured

Answer 7

primers

Question 8

part of PCR reaction mix: adds G, C, A, and Ts to make copies of DNA

Answer 8

polymerase (Taq polymerase)

Question 9

why does DNA and polymerase have to be thermostable in PCR?

Answer 9

because hi heat is used to denature DNA

Question 10

isolated from thermis aquaticis (thermophilic bacterium); stable at hi temps of PCR

Answer 10

Taq polymerase

Question 11

part of PCR reaction mix: G, C, A, and Ts

Answer 11

deoxyribonucleotide triphosphates (dNTPs)

Question 12

3 repeated steps of PCR:

Answer 12

1) denaturing
2) annealing
3) extension

Question 13

repeated step of PCR: target DNA denatured with heat; H bonds are released to get single strands of DNA

Answer 13

denaturing

Question 14

repeated step of PCR: primers bind to target DNA (primer for beginning of segment + one for end)

Answer 14

annealing

Question 15

repeated step of PCR: copies of target DNA are synthesized; ATCGs are added to complementary sides of DNA using Taq polymerase

Answer 15

extension

Question 16

T/F: temperatures go up and down in PCR

Answer 16

true

Question 17

PCR protocol of temperatures + times:

Answer 17

1) 95 °C for 1 min
2) 30 cycl3s of
- 95 °C for 30 seconds
- 55 °C for 1 minute
- 72 °C for 1 min
3) hold at 4 °C

Question 18

a ________ is used to complete PCR cycles

Answer 18

thermocycler

Question 19

why are so many cycles done in PCR?

Answer 19

to get a LOT of copies of DNA

Question 20

Lambda d gal is a
a) bacteriophage that carries the genes for galactose utilization
b) defective bacteriophage that cannot make mature phage after infection
c) possible product of generalized transduction
d) all of the above
e) A and B only

Answer 20

e) A and B only

Question 21

in PCR, primers are going in _________ direction

Answer 21

different (opposite)

Question 22

since primer are coming from opposite direction, the _______ of DNA is amplified

Answer 22

middle

Question 23

the “d” in Lambda d gal stands for ________

Answer 23

defective

Question 24

determines what part of the DNA is to be amplified in PCR

Answer 24

primers

Question 25

copies of target sequence in PCR increases _________ for each cycle

Answer 25

exponentially

Question 26

of copies of target sequence for PCR cycle 1:

Answer 26

2

Question 27

of copies of target sequence for PCR cycle 2:

Answer 27

4

Question 28

of copies of target sequence for PCR cycle 3:

Answer 28

8

Question 29

of copies of target sequence for PCR cycle 20:

Answer 29

10^8

Question 30

isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated; good to have a lot of copies; RECOMBINANT DNA

Answer 30

molecular cloning

Question 31

3 main steps of molecular cloning:

Answer 31

1) isolation and fragmentation of source DNA (cut) 2) insertion of DNA fragment into cloning vector 3) introduction of cloned DNA into host organism

Question 32

enzymes found in bacteria; used as "molecular scissors;" VERY specific; used to cut host vector as well

Answer 32

restriction endonucleases

Question 33

different kinds of cuts/end from restriction endonuclease:

Answer 33

- sticky - blunt

Question 34

staggered cut; more EFFICIENT; has an overhang

Answer 34

sticky end

Question 35

straight cut of DNA; less efficient

Answer 35

blunt end

Question 36

EcoRI cuts _______ ends and EcoRV cuts ________ ends

Answer 36

sticky; blunt

Question 37

"molcular glue"

Answer 37

ligase

Question 38

T/F: you only use 1 restriction enzyme to cut DNA in molecular cloning

Answer 38

false (can use more)

Question 39

plasmids as cloning vectors are easy to ________ from bacteria

Answer 39

purify (plasmid extraction)

Question 40

plasmids are _______ in size

Answer 40

small

Question 41

plasmids are smaller than __________; useful to get over cell wall and membrane

Answer 41

chromosome

Question 42

plasmids have an independent _________ _____ ________

Answer 42

origin of replication (ori)

Question 43

plasmids have a ________ copy number

Answer 43

multiple

Question 44

plasmids have presence of _________ _______

Answer 44

selectable markers (ex: antibiotic resistance)

Question 45

example of a selectable marker in plasmids

Answer 45

antibiotic resistance

Question 46

selectable markers can _______ others

Answer 46

disrupt

Question 47

lacZ disrupts _______ and its beta galactydase gene

Answer 47

lacC

Question 48

MCS =

Answer 48

multiple cloning site

Question 49

part of lac operon; gives bacteria ability to use lactose as a carbon source

Answer 49

lacZ

Question 50

plasmids have unique ______ _______

Answer 50

cloning sites

Question 51

cloning sites are also known as _________

Answer 51

polylinker

Question 52

what region on the plasmid is the polylinker/unique cloning site?

Answer 52

MCS

Question 53

color of colony that took plasmid but gene of interest did NOt go in

Answer 53

blue

Question 54

color of colony with gene of interest

Answer 54

white