Biotechnology Flashcards
What is PCR, what does it stand for
PCR
polymerase chain replication
DNA amplification
process creates many copies of specific DNA seq
What are the steps of PCR and what happens
also temperature
PCR
Denaturation - temp increased to seperate DNA stands (95 C )
Annealing - temp decreaseed so premade primers can attach (50 - 65C)
extension - polymerase extends primers (72 C )
How many times do you need to do PCR to get correctly sized target seq
PCR
3 times
How many times is PCR usually run
PCR
30 times to get the right amt of DNA
2^30 copies
Natural vs PCR DNA rep process
PCR
Unwinding
- Nat: helicase, start at ori
- PCR - DNA template, heat
Priming
- Nat: RNA primer, primase
- PCR: premade DNA primer (x2)
ELongation
- Nat: DNAP, Nucleotides
- PCR: DNAP Taq, nucleotides
Termination
-Nat: end of bubble/chromosome
- PCR: end of seq, change in temp
WHat is Taq polymerase and why is it important
PCR
DNAP from bacteria that live in hot springs
normal human DNAP would denature in high temp of PCR steps
makes process faster
Why can DNA primers be used insteaf of RNA
PCR
primers in PCR are made in lab, not with DNAP
more temperature stable, less likely to degrade
advantages vs disadvantages of this in vitro method
PCR
adv -sensitive
disadv - in vivo is faster
What is the purpose of Gel electrophoresis
Gel electro
seperation of nuecleic acids based on size, by moving them through a gel medium using electric current
can also be done with proteins
direction that DNA will travel in gel
Gel electro
DNA is negative
will move towards positive charged end
What is liquid buffer
Gel electro
provides medium for electric current
contains ions
prevents gel from overheating and drying out
gel sits in it
What are the 2 types of gel medium
Gel electro
agarose - seaweed, low res
polyacrylamide - artificial polymer, high res
polyacrylamid also workds for proteins
What is the purpose the DNA ladder
Gel electro
to ocmpare DNA strands with, has known DNA sizes
A mixture of DNA fragments of known sizes that increase by regular intervals
What is purpose of loading dye
Gel electro
used to track the DNA because its invisibel
Dye has certain weight to it, tells you when to turn off the gel so DNA doesn’t run off
makes the DNA heavier
How does ethium bromide stain DNA
also why is it useful
gel electro
binds to DNA
glows under UV light
is intercalating agent, gets stuck in between rungs of DNA’
is carcinogenic
WHat does it mean when the band is thick/bright
gel electro
there is a lot of DNA that is that length
use leading edge of band to determine size
What is the function of restriciton enzymes in bacterial cells
restrict enz
used in bacteria as immune system
protects from phages - cuts their DNA
makes the DNA harmless
What are restriction enzymes
restrict enz
endonuclease is example, like scissors
breaks phosphodiester bonds in DNA
causes sticky ends
recognizes specific DNA seqs
what are restriction sites
restrict enz
site recognized and cut by restriction enzymes
4-8 bp in length
is palindromic
ex
5’ G A A T T C 3’
3’ C T T A A G 5’
what are restriction fragments
restrict enz
pieces of DNA created by restriction enzymes
What are the 2 types of end restriction enzymes make
restrict enz
sticky ends - single stranded end of restriction fragment
blunt ends - when both strands are same length, no single stranded seg
where do restriction enz cut
restrict enz
on restriction sites
cuts at the same site on both strands
ex
5’ G | A A T T C 3’
3’ C T T A A | G 5’
What does it mean when there is a 5’ or 3’ overhang
restrict enz
5’ - when the 5’ end has sticky end
3’ - when 3’ end has the sticky end
How do you name restriction enz
restrict enz
First letter is first letter of genus
2,3 letter is first 2 letters of species
Capital letter(optional) - the strain type
Roman numeral - what order it was discoverd in
get restrict enz name
Bacillus amyloliquefaciens, strain H, 5th endonuclease identified
restrict enz
BamHV
get restrict enz name
Nocardia otitidis, 3rd endonuclease identified
restrict enz
NotIII
What is recombinant DNA
restrict enz
genes from two different sources (often different species) combined into one molecule
Why are restrict enz important in recombinant DNA tech
restrict enz
any DNA cut by same type of rest enzyme will have same sticky ends
allows DNA from very diff species to be combined
What are the steps in recombinant DNA tech
complete 5
restrict enz
- rest enz recognizes its specific palindrome
- rest enz cuts at sites
- add diff fragment cut by same enzyme (means sticky ends are same)
- frag attaches by complimentary base pairing
- DNA ligase closes bonds
what is plasmid
restrict enz
DNA from bacteria where gene of interest is being added
is circular
What are steps in Forming Recombinant DNA
restrict enz
- rest enz cuts gene of interest and plasmid
- gene of of interest added due to matching sticky ends
- ligase of the ends
What is purpose of CRISPR
CRISPR
exists naturally as immune system of bacteria
recognizes foreign DNA and will ut the DNA
Describe CRISPR locus in DNA
describe the 2 parts
CRISPR
has 2 parts
Spacers - saved DNA memories of previous virus infections
repeats - regularly spaceed, short, identical, palindromic
What does CRISPR stand for
CRISPR
clustered regularly interspaced short palindromic repeats
What is Cas genes
CRISPR
CRISPR associated genes
makes the CRISPR locus
carries out CRISPR mech
what are the 2 steps in CRISPR/CAS9 mech and their substeps
1.4, 2.2
A. Immunization
1.get foreign DNA - if bact survives infection
2.transcribe to RNA - into pre- crRNA
3.process/cut RNA - one spacer per cut, makes crRNA
4.encorperate into CAS9 protein - to be used to for recognition
B.adaptive immune system
1. target of DNA by crRNA
2. inactivation
when are repeaters made
CRISPR
when a new spacer is inserted, a repater is also added right after
What happens in Transcription to RNA step in CRISPR mech
Step A2
what is the product called
CRISPR
CRISPR locus is transcribed
created RNA called pre-crRNA
repeater seq make hairpin loops