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1
Q

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
Q

get restrict enz name

Bacillus amyloliquefaciens, strain H, 5th endonuclease identified

restrict enz

A

BamHV

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26
Q

get restrict enz name

Nocardia otitidis, 3rd endonuclease identified

restrict enz

A

NotIII

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27
Q

What is recombinant DNA

restrict enz

A

genes from two different sources (often different species) combined into one molecule

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28
Q

Why are restrict enz important in recombinant DNA tech

restrict enz

A

any DNA cut by same type of rest enzyme will have same sticky ends
allows DNA from very diff species to be combined

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29
Q

What are the steps in recombinant DNA tech

complete 5

restrict enz

A
  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
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30
Q

what is plasmid

restrict enz

A

DNA from bacteria where gene of interest is being added
is circular

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31
Q

What are steps in Forming Recombinant DNA

restrict enz

A
  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
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32
Q

What is purpose of CRISPR

CRISPR

A

exists naturally as immune system of bacteria
recognizes foreign DNA and will ut the DNA

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33
Q

Describe CRISPR locus in DNA

describe the 2 parts

CRISPR

A

has 2 parts
Spacers - saved DNA memories of previous virus infections
repeats - regularly spaceed, short, identical, palindromic

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34
Q

What does CRISPR stand for

CRISPR

A

clustered regularly interspaced short palindromic repeats

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35
Q

What is Cas genes

CRISPR

A

CRISPR associated genes
makes the CRISPR locus
carries out CRISPR mech

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36
Q

what are the 2 steps in CRISPR/CAS9 mech and their substeps

1.4, 2.2

A

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

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37
Q

when are repeaters made

CRISPR

A

when a new spacer is inserted, a repater is also added right after

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38
Q

What happens in Transcription to RNA step in CRISPR mech

Step A2
what is the product called

CRISPR

A

CRISPR locus is transcribed
created RNA called pre-crRNA
repeater seq make hairpin loops

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39
Q

when is crRNA made, what is it

CRISPR

A

third step of immunization part of CRISPR
pre-crRNA is cut into segs so that each contain i repeat and 1 spacer

40
Q

How do the CAS9 proteins help

CRISPR

A

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
Q

Why is CRSPR/CAS9 useful in gene editing

CRISPR

A

Manipulate the crRNA sequence to target whatever you want and Cas9 will cut it
makes gene editing precise and efficient

42
Q

What is gRNA

CRISPR

A

used in gene editing
RNA seq that is complementary to DNA seq of interest
called guide RNA
guides CAS9 to target site

43
Q

What is PAM

CRISPR

A

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
Q

What are the 2 types of repairs sys after CAS9 in human body

CRISPR

A

HDR: Homology directed repair
NHEJ: Non-homologous end joining

45
Q

Describe HDR method of DNA repair

CRISPR

A

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
Q

What is gene cloning describe

gene clone

A

making multi copies of 1 gene using in vivo amplification

47
Q

what is plasmid and its advantages

gene clone

A

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
Q

What are the steps in gene cloning

gene clone

A
  1. forming recombinant DNA
  2. transformaiton followed by many cell divisions
  3. selection
49
Q

What is a cloning vector

and components

gene clone

A

An artificially manipulated plasmid into which the gene of interest is introduced
Vector contains:
Ori
promoter
restriction sites / cloning site

50
Q

Cloning vector components

gene clone

A

1.Ori - allows plasmid to replicated in cell
2.promoter
3.restriction/cloning sites - where gene of interest is inserted , promoter is upstream

51
Q

what are the 2 mthods of transformation

gene clone

A

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
Q

What are the 3 possible outcomes from transformation

gene clone

A
  1. just gene of interest, no vector
  2. empty vector
  3. recombinant
53
Q

How Is the cell checked for if the transformation was successful

gene clone

A

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
Q

What does X-gal do in selection process

gene clone

A

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
Q

What does ampicillan do in selectrion proces s

gene clone

A

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
Q

What colour would a product with just the insert DNA be in x-Gal+ ampicillan medium

gene clone

A

would be white in x-gal - no lacZ
would not grow in ampicillan - no ampR
would have no growth

57
Q

What would a blue alive product mean if you put the bacteria in ampicillan + X Gal medium

gene clone

A

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
Q

What is a genome

DNA seq

A

an organisms’ complete set of DNA
Human genome has about 3 billion base pair

59
Q

Method used in DNA sequencing in Celara vs human genome project

DNA seq

A

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
Q

What is fredrick sangers DNA seq method

DNA seq

A

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
Q

dideoxyribonucleotides vs deoxyribonucleotides

also what is importance of difference

DNA seq

A

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
Q

How do you know the 3’end and 5’ end in gel electro

DNA seq

A

the ones that go that farthest are on the 5’ end, this is where elongation starts,
segs are the shortest

63
Q

modern sequencing vs sanger method

DNA seq

A

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
Q

What does an overlapping bump of diff colours mean in modern DNA seq

DNA seq

A

means that there is a mutation there
2 possible bases have been recorded in same spot

65
Q

true or false : lot more noncoding regions than coding regions in human DNA

DNA fingerp

A

true, r

66
Q

True or false

DNA fingerp looks at genetic seq to differentiate between indiv

DNA fingerp

A

false, looks at the length in polymorphic regions not seq of bases

67
Q

What are the 3 types of noncoding seq

DNA fingerp

A

introns
highly repetitive DNA (has 3 subunits)
regulatory seq

68
Q

What are the 3 types of highly repetitive DNA

DNA fingerp

A

tandomly repetitive DNA
telomeres
interspersed repetitive DNA (transposon)

69
Q

what is VNTRs vs STR

DNA fingerp

A

Variable number tandem repeats - short (10-60 ) identical repeating base pairs
Short tandem repeats - 4 bases long also repeating in tandem

70
Q

What does polymorphic mean in terms of VNTRs

DNA fingerp

A

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
Q

2 applications of VNTR

DNA fingerp

A

DNA fingerprinting - between people
studying evolution - between species

72
Q

applications DNA fingerprinting other than id criminals

DNA fingerp

A

paternity tests
food tessting
immigration disputes
id a corpse

73
Q

Why are noncoding regions used in DNA fingerprinting instead of coding

DNA fingerp

A

coding reagions are too similar between things of same species

74
Q

why are tandem reapeats used in DNA finerprinting insteat of interspersed repeats

DNA fingerp

A

you can count how many repeats there are in tandem repeats, varies between person to person
interspersed is harder to compare

75
Q

How is PCR used in DNA fingerprinting

DNA fingerp

A

used to amplify the DNA so it can be used in gel electrophoresis

76
Q

how are STRs isolated to be used in gel electrophoresis

DNA fingerp

A

they are targeted with seq specific primers and amplified using PCR to be used in gel elctro

77
Q

allelic choices in populatin vs person

DNA fingerp

A

in population there could be many lengths of the same allels, more than 2
a person of the population has only 2 choices

78
Q

What did miesher do

DNA exp

A

discovered DNA
isolated nuclei

79
Q

R cells vs S cells

DNA exp

A

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
Q

what did griffith do

purpose+ exp+concl

DNA exp

A

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
Q

why did the dead S cells + Rcells kill the mice

DNA exp

A

the R cells were able to replicate using the DNA of the S cells
allowed them to make shells
saw DNA transformation

82
Q

what was the hammerling exp

purpose+ exp+concl

DNA exp

A

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
Q

what was Avery et al. exp

DNA exp

A

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
Q

What was hershey chase exp

DNA exp

A

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
Q

What were the componets of test tube after centrifuge in hershey chase exp

DNA exp

A

supernatent - has viruses - radioactive if protein was radio
pellet - has bacteria - radio active if viral DNA was radio

86
Q

Why did they use radio sulpher and phos for protein and DNA

DNA exp

A

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
Q

What was beadle and Tatums exp for, what was it

DNA exp

A

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
Q

Why did beadle + tatum use bread mold

DNA exp

A

short life cycle, replicates fast
needs minimal external food
can make most nutrients itself
can grow in minimal media+

89
Q

What are the compoents that make up the minimal media, complete and supplemented media

DNA exp

A

complete - has salt, sugar, all vitamins and amino acids
minimal- just salt sugar , biotin
sumplimented - minimal + one type of vitamin or amino acid

90
Q

what are the 3 steps in beadle and tatum exp

DNA exp

A
  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
Q

what were conclustions of beadle and tatum first exp

DNA exp

A

were able to see that mutations in DNA caused change in enzymic pathway, meaning genes are responsible for enz production

92
Q

what was second beadle tatum exp

DNA exp

A

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
Q

What does it mean when mold grows in seond test tube and third but not first

beadle tatum exp #2

DNA exp

A

enzyme used between the first and second step of pathway cannot be made

94
Q

how does beadle and taums exp relate to one gene one enzyme hypthesis

DNA exp

A

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
Q

why is one gene one enzyme theory inaccurate

DNA exp

A

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
Q

in what order were the DNA experiments done

DNA exp

A

griffith
hammerling
avery
hershey chase

Biology 12 (8 decks)

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