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Biology 12 > Biotechnology > Flashcards

Biotechnology Flashcards

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1
Q

f

What is PCR, what does it stand for

PCR

A

polymerase chain replication
DNA amplification
process creates many copies of specific DNA seq

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2
Q

What are the steps of PCR and what happens

also temperature

PCR

A

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 )

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3
Q

How many times do you need to do PCR to get correctly sized target seq

PCR

A

3 times

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4
Q

How many times is PCR usually run

PCR

A

30 times to get the right amt of DNA
2^30 copies

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5
Q

Natural vs PCR DNA rep process

PCR

A

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

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6
Q

WHat is Taq polymerase and why is it important

PCR

A

DNAP from bacteria that live in hot springs
normal human DNAP would denature in high temp of PCR steps
makes process faster

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7
Q

Why can DNA primers be used insteaf of RNA

PCR

A

primers in PCR are made in lab, not with DNAP
more temperature stable, less likely to degrade

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8
Q

advantages vs disadvantages of this in vitro method

PCR

A

adv -sensitive
disadv - in vivo is faster

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9
Q

What is the purpose of Gel electrophoresis

Gel electro

A

seperation of nuecleic acids based on size, by moving them through a gel medium using electric current
can also be done with proteins

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10
Q

direction that DNA will travel in gel

Gel electro

A

DNA is negative
will move towards positive charged end

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11
Q

What is liquid buffer

Gel electro

A

provides medium for electric current
contains ions
prevents gel from overheating and drying out
gel sits in it

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12
Q

What are the 2 types of gel medium

Gel electro

A

agarose - seaweed, low res
polyacrylamide - artificial polymer, high res

polyacrylamid also workds for proteins

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13
Q

What is the purpose the DNA ladder

Gel electro

A

to ocmpare DNA strands with, has known DNA sizes
A mixture of DNA fragments of known sizes that increase by regular intervals

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14
Q

What is purpose of loading dye

Gel electro

A

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

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15
Q

How does ethium bromide stain DNA

also why is it useful

gel electro

A

binds to DNA
glows under UV light
is intercalating agent, gets stuck in between rungs of DNA’
is carcinogenic

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16
Q

WHat does it mean when the band is thick/bright

gel electro

A

there is a lot of DNA that is that length
use leading edge of band to determine size

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17
Q

What is the function of restriciton enzymes in bacterial cells

restrict enz

A

used in bacteria as immune system
protects from phages - cuts their DNA
makes the DNA harmless

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18
Q

What are restriction enzymes

restrict enz

A

endonuclease is example, like scissors
breaks phosphodiester bonds in DNA
causes sticky ends
recognizes specific DNA seqs

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19
Q

what are restriction sites

restrict enz

A

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’

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20
Q

what are restriction fragments

restrict enz

A

pieces of DNA created by restriction enzymes

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21
Q

What are the 2 types of end restriction enzymes make

restrict enz

A

sticky ends - single stranded end of restriction fragment
blunt ends - when both strands are same length, no single stranded seg

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22
Q

where do restriction enz cut

restrict enz

A

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’

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23
Q

What does it mean when there is a 5’ or 3’ overhang

restrict enz

A

5’ - when the 5’ end has sticky end
3’ - when 3’ end has the sticky end

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24
Q

How do you name restriction enz

restrict enz

A

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

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25
# get restrict enz name Bacillus amyloliquefaciens, strain H, 5th endonuclease identified ## Footnote restrict enz
BamHV
26
# get restrict enz name Nocardia otitidis, 3rd endonuclease identified ## Footnote restrict enz
NotIII
27
What is recombinant DNA ## Footnote restrict enz
genes from two different sources (often different species) combined into one molecule
28
Why are restrict enz important in recombinant DNA tech ## Footnote 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
29
What are the steps in recombinant DNA tech | complete 5 ## Footnote restrict enz
1. rest enz recognizes its specific palindrome 2. rest enz cuts at sites 3. add diff fragment cut by same enzyme (means sticky ends are same) 4. frag attaches by complimentary base pairing 5. DNA ligase closes bonds
30
what is plasmid ## Footnote restrict enz
DNA from bacteria where gene of interest is being added is circular
31
What are steps in Forming Recombinant DNA ## Footnote restrict enz
1. rest enz cuts gene of interest and plasmid 2. gene of of interest added due to matching sticky ends 3. ligase of the ends
32
What is purpose of CRISPR ## Footnote CRISPR
exists naturally as immune system of bacteria recognizes foreign DNA and will ut the DNA
33
Describe CRISPR locus in DNA | describe the 2 parts ## Footnote CRISPR
has 2 parts Spacers - saved DNA memories of previous virus infections repeats - regularly spaceed, short, identical, palindromic
34
What does CRISPR stand for ## Footnote CRISPR
clustered regularly interspaced short palindromic repeats
35
What is Cas genes ## Footnote CRISPR
CRISPR associated genes makes the CRISPR locus carries out CRISPR mech
36
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
37
when are repeaters made ## Footnote CRISPR
when a new spacer is inserted, a repater is also added right after
38
What happens in Transcription to RNA step in CRISPR mech | Step A2 what is the product called ## Footnote CRISPR
CRISPR locus is transcribed created RNA called pre-crRNA repeater seq make hairpin loops
39
when is crRNA made, what is it ## Footnote CRISPR
third step of immunization part of CRISPR pre-crRNA is cut into segs so that each contain i repeat and 1 spacer
40
How do the CAS9 proteins help ## Footnote 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
41
Why is CRSPR/CAS9 useful in gene editing ## Footnote CRISPR
Manipulate the crRNA sequence to target whatever you want and Cas9 will cut it makes gene editing precise and efficient
42
What is gRNA ## Footnote CRISPR
used in gene editing RNA seq that is complementary to DNA seq of interest called guide RNA guides CAS9 to target site
43
What is PAM ## Footnote 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
44
What are the 2 types of repairs sys after CAS9 in human body ## Footnote CRISPR
HDR: Homology directed repair NHEJ: Non-homologous end joining
45
Describe HDR method of DNA repair ## Footnote 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
46
What is gene cloning describe ## Footnote gene clone
making multi copies of 1 gene using in vivo amplification
47
what is plasmid and its advantages ## Footnote 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
48
What are the steps in gene cloning ## Footnote gene clone
1. forming recombinant DNA 2. transformaiton followed by many cell divisions 3. selection
49
What is a cloning vector | and components ## Footnote gene clone
An artificially manipulated plasmid into which the gene of interest is introduced Vector contains: Ori promoter restriction sites / cloning site
50
Cloning vector components ## Footnote 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
51
what are the 2 mthods of transformation ## Footnote 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
52
What are the 3 possible outcomes from transformation ## Footnote gene clone
1. just gene of interest, no vector 2. empty vector 3. recombinant
53
How Is the cell checked for if the transformation was successful ## Footnote 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
54
What does X-gal do in selection process ## Footnote 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
55
What does ampicillan do in selectrion proces s ## Footnote 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
56
What colour would a product with just the insert DNA be in x-Gal+ ampicillan medium ## Footnote gene clone
would be white in x-gal - no lacZ would not grow in ampicillan - no ampR would have no growth
57
What would a blue alive product mean if you put the bacteria in ampicillan + X Gal medium ## Footnote 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
58
What is a genome ## Footnote DNA seq
an organisms’ complete set of DNA Human genome has about 3 billion base pair
59
Method used in DNA sequencing in Celara vs human genome project ## Footnote 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
60
What is fredrick sangers DNA seq method ## Footnote 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
61
dideoxyribonucleotides vs deoxyribonucleotides | also what is importance of difference ## Footnote 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
62
How do you know the 3'end and 5' end in gel electro ## Footnote DNA seq
the ones that go that farthest are on the 5' end, this is where elongation starts, segs are the shortest
63
modern sequencing vs sanger method ## Footnote 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
64
What does an overlapping bump of diff colours mean in modern DNA seq ## Footnote DNA seq
means that there is a mutation there 2 possible bases have been recorded in same spot
65
true or false : lot more noncoding regions than coding regions in human DNA ## Footnote DNA fingerp
true, r
66
# True or false DNA fingerp looks at genetic seq to differentiate between indiv ## Footnote DNA fingerp
false, looks at the length in polymorphic regions not seq of bases
67
What are the 3 types of noncoding seq ## Footnote DNA fingerp
introns highly repetitive DNA (has 3 subunits) regulatory seq
68
What are the 3 types of highly repetitive DNA ## Footnote DNA fingerp
tandomly repetitive DNA telomeres interspersed repetitive DNA (transposon)
69
what is VNTRs vs STR ## Footnote DNA fingerp
Variable number tandem repeats - short (10-60 ) identical repeating base pairs Short tandem repeats - 4 bases long also repeating in tandem
70
What does polymorphic mean in terms of VNTRs ## Footnote 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
71
2 applications of VNTR ## Footnote DNA fingerp
DNA fingerprinting - between people studying evolution - between species
72
applications DNA fingerprinting other than id criminals ## Footnote DNA fingerp
paternity tests food tessting immigration disputes id a corpse
73
Why are noncoding regions used in DNA fingerprinting instead of coding ## Footnote DNA fingerp
coding reagions are too similar between things of same species
74
why are tandem reapeats used in DNA finerprinting insteat of interspersed repeats ## Footnote DNA fingerp
you can count how many repeats there are in tandem repeats, varies between person to person interspersed is harder to compare
75
How is PCR used in DNA fingerprinting ## Footnote DNA fingerp
used to amplify the DNA so it can be used in gel electrophoresis
76
how are STRs isolated to be used in gel electrophoresis ## Footnote DNA fingerp
they are targeted with seq specific primers and amplified using PCR to be used in gel elctro
77
allelic choices in populatin vs person ## Footnote 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
78
What did miesher do ## Footnote DNA exp
discovered DNA isolated nuclei
79
R cells vs S cells ## Footnote 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,
80
what did griffith do | purpose+ exp+concl ## Footnote 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
81
why did the dead S cells + Rcells kill the mice ## Footnote DNA exp
the R cells were able to replicate using the DNA of the S cells allowed them to make shells saw DNA transformation
82
what was the hammerling exp | purpose+ exp+concl ## Footnote 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
83
what was Avery et al. exp ## Footnote 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
84
What was hershey chase exp ## Footnote 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
85
What were the componets of test tube after centrifuge in hershey chase exp ## Footnote DNA exp
supernatent - has viruses - radioactive if protein was radio pellet - has bacteria - radio active if viral DNA was radio
86
Why did they use radio sulpher and phos for protein and DNA ## Footnote 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
87
What was beadle and Tatums exp for, what was it ## Footnote 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
88
Why did beadle + tatum use bread mold ## Footnote DNA exp
short life cycle, replicates fast needs minimal external food can make most nutrients itself can grow in minimal media+
89
What are the compoents that make up the minimal media, complete and supplemented media ## Footnote 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
90
what are the 3 steps in beadle and tatum exp ## Footnote DNA exp
1. Xray bact - cause mutations in DNA 2. isolate mutents - those that could grow in complete, not minimal media 3. identify mutents - see which type of suplimented media it could grow in, means it could not make it by itself
91
what were conclustions of beadle and tatum first exp ## Footnote DNA exp
were able to see that mutations in DNA caused change in enzymic pathway, meaning genes are responsible for enz production
92
what was second beadle tatum exp ## Footnote 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
93
What does it mean when mold grows in seond test tube and third but not first | beadle tatum exp #2 ## Footnote DNA exp
enzyme used between the first and second step of pathway cannot be made
94
how does beadle and taums exp relate to one gene one enzyme hypthesis ## Footnote 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
95
why is one gene one enzyme theory inaccurate ## Footnote 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
96
in what order were the DNA experiments done ## Footnote DNA exp
griffith hammerling avery hershey chase

Biology 12 (10 decks)

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