Biotechnology Flashcards
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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
when is crRNA made, what is it
CRISPR
third step of immunization part of CRISPR
pre-crRNA is cut into segs so that each contain i repeat and 1 spacer
How do the CAS9 proteins help
CRISPR
they contain crRNA
if infected by same virus
- crRNA will match with complimentary DNA in virus
- will activate endonuclease abilities of CAS9
- will make double stranded blunt cut and destroy invading virus DNA
Why is CRSPR/CAS9 useful in gene editing
CRISPR
Manipulate the crRNA sequence to target whatever you want and Cas9 will cut it
makes gene editing precise and efficient
What is gRNA
CRISPR
used in gene editing
RNA seq that is complementary to DNA seq of interest
called guide RNA
guides CAS9 to target site
What is PAM
CRISPR
Protospacer Adjacent Motif
seq 5’-NGG-3 that is common in human genome
where CAS9 binds, makes CAS9 unwind DNA
cut from CAS9 happens upstream of PAM
What are the 2 types of repairs sys after CAS9 in human body
CRISPR
HDR: Homology directed repair
NHEJ: Non-homologous end joining
Describe HDR method of DNA repair
CRISPR
repair by insertion
insert desired DNA seq flanked by homologous DNA seq
when DNA is fixed it uses the DNA provided as base and encorperates it
What is gene cloning describe
gene clone
making multi copies of 1 gene using in vivo amplification
what is plasmid and its advantages
gene clone
small number of genes
not part of bact chromosme but can be incorperated into it
not required for bact to survive
increase genetic variation
What are the steps in gene cloning
gene clone
- forming recombinant DNA
- transformaiton followed by many cell divisions
- selection
What is a cloning vector
and components
gene clone
An artificially manipulated plasmid into which the gene of interest is introduced
Vector contains:
Ori
promoter
restriction sites / cloning site
Cloning vector components
gene clone
1.Ori - allows plasmid to replicated in cell
2.promoter
3.restriction/cloning sites - where gene of interest is inserted , promoter is upstream
what are the 2 mthods of transformation
gene clone
natural - bacataeria naturally takes in DNA it is given
artificial- bacteria takes in DNA because cell wall has been make permeable by us
Using chemicals or electricity to make holes
What are the 3 possible outcomes from transformation
gene clone
- just gene of interest, no vector
- empty vector
- recombinant
How Is the cell checked for if the transformation was successful
gene clone
place into diff mediums to replicate changes colour depending on components
x-gal - if cells are blue then vector is empty, if white then recombinant
ampR drug - if white then vector is present, ampR is drug resist gene in bact
proper recombinants should be white
What does X-gal do in selection process
gene clone
second step in selection process
lacZ makes enzymes that make it blue
insert DNA into lacZ so it does not turn blue in x-Gal
white means that lacZ not working - correct transformation
blue - lacZ working, no inserted DNA
What does ampicillan do in selectrion proces s
gene clone
plasmid usually resistant to ampicillan
only take cells that can live in ampicillan, means they have vector
vector would have the insert DNA or not
move to x-Gal to confirm
What colour would a product with just the insert DNA be in x-Gal+ ampicillan medium
gene clone
would be white in x-gal - no lacZ
would not grow in ampicillan - no ampR
would have no growth
What would a blue alive product mean if you put the bacteria in ampicillan + X Gal medium
gene clone
would have vector but no insert DNA
insert DNA would make it white in x-Gal
resistant to ampR so it has vector
would grow and be blue
What is a genome
DNA seq
an organisms’ complete set of DNA
Human genome has about 3 billion base pair
Method used in DNA sequencing in Celara vs human genome project
DNA seq
celara - randomly breaking up DNA, reconstructing it by looking for parts that overlap
HGP - same as celara but markers are used at regular intervals so reassebling is easier
What is fredrick sangers DNA seq method
DNA seq
developed dideoxy termination seq method
setup 4 diff test tubes
each test tube has all 4 dNTP
also includes 1 type of ddNTP
new strands are made with different lengths depending on when ddNTP was incorperated
use gel electrophoresis to determine order of bases
dideoxyribonucleotides vs deoxyribonucleotides
also what is importance of difference
DNA seq
ddNTP - missing O in third postion and second
dNTP - missing O in just sescond pos
ddNTP causes elongation to stop, it does not have O for phosphodiester bond to happen with next nucleotide
both have phosphate, sugar and base
How do you know the 3’end and 5’ end in gel electro
DNA seq
the ones that go that farthest are on the 5’ end, this is where elongation starts,
segs are the shortest
modern sequencing vs sanger method
DNA seq
the different ddNTPs are dyes differently
allows it to happen in same lane of gel electro and same test tube
look at colour of bands to tell the DNA seq
What does an overlapping bump of diff colours mean in modern DNA seq
DNA seq
means that there is a mutation there
2 possible bases have been recorded in same spot
true or false : lot more noncoding regions than coding regions in human DNA
DNA fingerp
true, r
True or false
DNA fingerp looks at genetic seq to differentiate between indiv
DNA fingerp
false, looks at the length in polymorphic regions not seq of bases
What are the 3 types of noncoding seq
DNA fingerp
introns
highly repetitive DNA (has 3 subunits)
regulatory seq
What are the 3 types of highly repetitive DNA
DNA fingerp
tandomly repetitive DNA
telomeres
interspersed repetitive DNA (transposon)
what is VNTRs vs STR
DNA fingerp
Variable number tandem repeats - short (10-60 ) identical repeating base pairs
Short tandem repeats - 4 bases long also repeating in tandem
What does polymorphic mean in terms of VNTRs
DNA fingerp
each person has 2 alleles for same gene
alelles of people can have different lengths
the number of times the sequence is repeated, can vary between individuals
2 applications of VNTR
DNA fingerp
DNA fingerprinting - between people
studying evolution - between species
applications DNA fingerprinting other than id criminals
DNA fingerp
paternity tests
food tessting
immigration disputes
id a corpse
Why are noncoding regions used in DNA fingerprinting instead of coding
DNA fingerp
coding reagions are too similar between things of same species
why are tandem reapeats used in DNA finerprinting insteat of interspersed repeats
DNA fingerp
you can count how many repeats there are in tandem repeats, varies between person to person
interspersed is harder to compare
How is PCR used in DNA fingerprinting
DNA fingerp
used to amplify the DNA so it can be used in gel electrophoresis
how are STRs isolated to be used in gel electrophoresis
DNA fingerp
they are targeted with seq specific primers and amplified using PCR to be used in gel elctro
allelic choices in populatin vs person
DNA fingerp
in population there could be many lengths of the same allels, more than 2
a person of the population has only 2 choices
What did miesher do
DNA exp
discovered DNA
isolated nuclei
R cells vs S cells
DNA exp
R cells - have no outer protection, rough, can be seen and destroyed by immune syste
S cells -bad, smooth, have outer shell, cannot be detected,
what did griffith do
purpose+ exp+concl
DNA exp
first of exp
relationship between r and s cells
infected diff types of cells into mice
R cells - mice alive
S Cells - mice dead
dead S cells - mice alice
R cells + S cells - mice dead
R cells can becom S cells, discovered transformation
why did the dead S cells + Rcells kill the mice
DNA exp
the R cells were able to replicate using the DNA of the S cells
allowed them to make shells
saw DNA transformation
what was the hammerling exp
purpose+ exp+concl
DNA exp
what part of organism is used to regenerate
used single cellular algae with head, stalk and foot
removed diff parts
head - regrow head
stalk + head - regrow
foot- dead
graphed 2 together, what grew was same type as foot
concluded that nucleus and rhizoid were location of hereditary info
what was Avery et al. exp
DNA exp
What was starting component in translation+transcription
kill S cells
add in enzymes that would destroy proteins, RNA, DNA
add R cells,
see which test tubes make new S cells
test tube without DNA made no S cells
concluded that DNA is needed to make things DNA needed for transcription
What was hershey chase exp
DNA exp
wanted to know what virus used to infect, DNA or protein
redioactive sulphur for labeling proteins
radioactive phos for DNA
allowed virus to inject into bact
centrifuge, saw which part was radio active
radio protein - virus was radioactiv
radio DNA - bact was radioactive
meant that DNA was transferred
What were the componets of test tube after centrifuge in hershey chase exp
DNA exp
supernatent - has viruses - radioactive if protein was radio
pellet - has bacteria - radio active if viral DNA was radio
Why did they use radio sulpher and phos for protein and DNA
DNA exp
had to use radioactive elements specific to the molecyle
some amino acids use sulphur
DNA uses phosphorus
if common element was used, results would be unclear
What was beadle and Tatums exp for, what was it
DNA exp
wanted to prove that genes were responsible for makeing enzymes
one gene one enzyme hypthesis
mutate the genes in mold
look for mutations that affected the mold’s ability to make essential nutrients
Why did beadle + tatum use bread mold
DNA exp
short life cycle, replicates fast
needs minimal external food
can make most nutrients itself
can grow in minimal media+
What are the compoents that make up the minimal media, complete and supplemented media
DNA exp
complete - has salt, sugar, all vitamins and amino acids
minimal- just salt sugar , biotin
sumplimented - minimal + one type of vitamin or amino acid
what are the 3 steps in beadle and tatum exp
DNA exp
- Xray bact - cause mutations in DNA
- isolate mutents - those that could grow in complete, not minimal media
- identify mutents - see which type of suplimented media it could grow in, means it could not make it by itself
what were conclustions of beadle and tatum first exp
DNA exp
were able to see that mutations in DNA caused change in enzymic pathway, meaning genes are responsible for enz production
what was second beadle tatum exp
DNA exp
looked specifically at arg pathway
Identified 3 classes of arg mutants by supplementing the different classes diff precursors of arg
wanted to dentify the step at which arg metabolic pathway was blocked by the mutation
What does it mean when mold grows in seond test tube and third but not first
beadle tatum exp #2
DNA exp
enzyme used between the first and second step of pathway cannot be made
how does beadle and taums exp relate to one gene one enzyme hypthesis
DNA exp
one gene one enz theory - gene dicates production of specific enz
each mutant was defective in single gene
caused issue in one enzyme in pathway
implied that metabolic diseases could be inherited
why is one gene one enzyme theory inaccurate
DNA exp
not all enzymes are proteis
protein may have subunits, coded by diff genes
genes may not produces stuff (UTR)
genes may produce many polypept due to splicing
in what order were the DNA experiments done
DNA exp
griffith
hammerling
avery
hershey chase
