PCR, Cloning, Plasmids (#3) Flashcards
synthesizes large quantities of DNA fragments
PCR
PCR =
polymerase chain reaction
application of PCR (4):
- species identification
- genotyping
- gene cloning
- forensics
who came up with PCR and when?
Karry Mullis in 1983
PCR reaction mix (5):
- target DNA
- primers
- thermostable DNA
- polyermase (Taq polymerase)
- deoxyribonucleotide triphosphates (dNTPs)
part of PCR reaction mix: need at least ONE piece of it
target DNA
short pieces of DNA made by genetic engineer; binds to complementary DNA after it’s denatured
primers
part of PCR reaction mix: adds G, C, A, and Ts to make copies of DNA
polymerase (Taq polymerase)
why does DNA and polymerase have to be thermostable in PCR?
because hi heat is used to denature DNA
isolated from thermis aquaticis (thermophilic bacterium); stable at hi temps of PCR
Taq polymerase
part of PCR reaction mix: G, C, A, and Ts
deoxyribonucleotide triphosphates (dNTPs)
3 repeated steps of PCR:
1) denaturing
2) annealing
3) extension
repeated step of PCR: target DNA denatured with heat; H bonds are released to get single strands of DNA
denaturing
repeated step of PCR: primers bind to target DNA (primer for beginning of segment + one for end)
annealing
repeated step of PCR: copies of target DNA are synthesized; ATCGs are added to complementary sides of DNA using Taq polymerase
extension
T/F: temperatures go up and down in PCR
true
PCR protocol of temperatures + times:
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
a ________ is used to complete PCR cycles
thermocycler
why are so many cycles done in PCR?
to get a LOT of copies of DNA
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
e) A and B only
in PCR, primers are going in _________ direction
different (opposite)
since primer are coming from opposite direction, the _______ of DNA is amplified
middle
the “d” in Lambda d gal stands for ________
defective
determines what part of the DNA is to be amplified in PCR
primers
copies of target sequence in PCR increases _________ for each cycle
exponentially
of copies of target sequence for PCR cycle 1:
2
of copies of target sequence for PCR cycle 2:
4
of copies of target sequence for PCR cycle 3:
8
of copies of target sequence for PCR cycle 20:
10^8
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
molecular cloning
3 main steps of molecular cloning:
1) isolation and fragmentation of source DNA (cut)
2) insertion of DNA fragment into cloning vector
3) introduction of cloned DNA into host organism
enzymes found in bacteria; used as “molecular scissors;” VERY specific; used to cut host vector as well
restriction endonucleases
different kinds of cuts/end from restriction endonuclease:
- sticky
- blunt
staggered cut; more EFFICIENT; has an overhang
sticky end
straight cut of DNA; less efficient
blunt end
EcoRI cuts _______ ends and EcoRV cuts ________ ends
sticky; blunt
“molcular glue”
ligase
T/F: you only use 1 restriction enzyme to cut DNA in molecular cloning
false (can use more)
plasmids as cloning vectors are easy to ________ from bacteria
purify (plasmid extraction)
plasmids are _______ in size
small
plasmids are smaller than __________; useful to get over cell wall and membrane
chromosome
plasmids have an independent _________ _____ ________
origin of replication (ori)
plasmids have a ________ copy number
multiple
plasmids have presence of _________ _______
selectable markers (ex: antibiotic resistance)
example of a selectable marker in plasmids
antibiotic resistance
selectable markers can _______ others
disrupt
lacZ disrupts _______ and its beta galactydase gene
lacC
MCS =
multiple cloning site
part of lac operon; gives bacteria ability to use lactose as a carbon source
lacZ
plasmids have unique ______ _______
cloning sites
cloning sites are also known as _________
polylinker
what region on the plasmid is the polylinker/unique cloning site?
MCS
color of colony that took plasmid but gene of interest did NOt go in
blue
color of colony with gene of interest
white
