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BIOL 200 > PL3 > Flashcards

PL3 Flashcards

1
Q

what are the fundamental techniques in molecular biology

A

polymerase chain reaction (PCR)
DNA sequencing

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2
Q

describe PCR (polymerase chain reaction)

A

amplifies a specific DNA sequence which can be part of a complex mixture
does not have to be purified

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3
Q

name 4 uses of PCR

A

sequencing (some approaches) - study sequence of that section
DNA cloning (isolating a particular gene) - to study it in more depth
detection of pathogens (like SARS-CoV-2)
gene editing (edit an amplified gene)

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4
Q

what does PCR generally depend on

A

knowing the nucleotide sequences at the ends of the region to be amplified

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5
Q

what does single reaction tube contain

A

DNA template (can be complex mixture, like total DNA from cell)
DNA polymerase that is stable at high temp (to make copies)
primers complementary to each end of the region to be amplified (oligionucleotides or oligos)
dNTPS (monomers to create new polymers)

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6
Q

does DNA need to be denatured for PCR and explain why or why not

A

yesss
heat destabilizes the double helix
need to be separated to use as templates
hot temp used - depends on G-C content - more G-C = higher temp since 3 H bonds (harder to break)

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7
Q

what is 3 step cycle of PCR

A

denaturation
annealing of primers
extension by DNA polymerase

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8
Q

how many repetitions are needed for PCR

A

20-40

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9
Q

describe denaturation (step of PCR)

A

temp around 95 or 98 degrees
hot enough regardless of G-C content
DNA strand separate

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10
Q

describe annealing of primers (step of PCR)

A

temp decreased to allow primers to base pair to complementary DNA template
anneals to each end of segment to be amplified
~50 degrees

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11
Q

describe extension by DNA polymerase (step of PCR)

A

polymerase extends primer to form nascent DNA strand
around 70 degrees
synthesis of new strand

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12
Q

describe thermal cycler

A

can rapidly change temp
used for PCR
hot plate with test tubes - fast at changing temp
before PCR was harder

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13
Q

describe the DNA primers (how they are made)

A

oligonucleotides are designed by computer to be complementary and specific to ends of the sequence to be amplified and commercially synthesized
cost very low <1dollar

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14
Q

which DNA polymerase is used for PCR

A

Taq polymerase

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15
Q

where does Taq polymerase come from

A

thermophilic bacterium (thermus aquaticus)

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16
Q

why is Taq polymerase good for PCR

A

thermally resistant so enzyme will not denature at high temp
cheap but no proofreading activity so it is best for amplifying short fragments
other enzymes with higher fidelity (accuracy) can also be used

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17
Q

what does exponential amplification allow for

A

a very sensitive detection of DNA sequence in the sample and enables purification of substantial amounts of a specific fragment for further use

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18
Q

why does PCR amplify DNA

A

each time you have more template strands

19
Q

can you keep doing PCR forever

A

primers and dNTPs can run out so no

20
Q

can primers anneal the wrong way

A

yessss s
can be created wrong
creates more off target effects

21
Q

what method can analyze DNA fragments produced by PCR

A

gel electrophoresis

22
Q

describe gel electrophoresis for PCR

A

solid like jelly = gel
they move inversely to length
separation based on size
if you wanted to amplify 1.5kb then you run electrophoresis and there should be a prominent band of DNA at 1.5kb - way to tell if it worked

23
Q

what is a method of DNA sequencing

A

dideoxy chain termination method of DNA sequencing
classical sanger sequencing

24
Q

is classic sanger sequencing still used

A

primary method from late 1970s to 2010 around
sometimes still used for verification of individual results

25
Q

what is in tubes for sanger sequencing

A

DNA polymerase (not done at high temp so can be any)
oligonucleotide primer (anneals to one end of sequence)
DNA template
dNTPs (100mM)
chain terminators

26
Q

how many tubes used in sanger sequencing

A

4

27
Q

name the chain terminators

A

ddATP (1mM) - red
ddGTP (1mM) - purple
ddTTP (1mM) - blue
ddCTP (1mM) - green
super low concentration compared to dNTPs

28
Q

describe ddNTP chain terminators

A

missing 3’OH - just H
we need 3’ OH for subsequent ntps to be incorporated
so when pol hits a ddNTP then synthesis of strand stops and polymerase falls off

29
Q

describe sanger sequencing - gen

A

add all components (primer, DNA pol, dNTPs and ddNTPs)
ex: using ddGTP
whenever it encounters a G - either dGTP or ddGTP will be added
if dGTP then pol will continue if ddGTP then pol
will fall off

30
Q

what can you see if sanger sequence ran on gel

A

bands corresponding to sequences
can show all possible lengths (4 reactions)
read sequences off gel

31
Q

what are the limitations of sanger sequencing

A

polymerase only runs for 300-500 nts and gels can only clearly resolve that much - separate individual sequencing reactions must be run to sequence large region
rate of sequence production limited by total number of reactions that can be performed at once - more expensive

32
Q

what is next generation sequencing (NGS)

A

technological breakthrough
methods were developed to allow a single sequencing instrument to carry out millions of sequencing reactions at same time
less money and more volume

33
Q

describe NGS technology

A

ligating the same linkers to a mixture of DNA fragments

34
Q

describe NGS - first part

A

DNA denatured and anneals to complementary primers anchored on solid support
PCR conducted (amplifies in a fixed spatial arrangement

35
Q

describe NGS - second part

A

double stranded DNA is cut and only one strand is sequenced
with fluorescently labeled dNTPs (diff colour for each base) - base pairs immediately adjacent to primer
imaging and removal of fluorophore takes place after each cycle
get pattern of where bases are

36
Q

describe an image of NGs

A

each point represents a diff DNA molecule being sequenced (cluster of DNA molecules close to each other due to PCR amplification)
procedure takes around a day and can yield 10^11 bases of sequence info

37
Q

describe assembly of whole genome sequences

A

getting a bunch of 100nts of info but want to know how they fit together

38
Q

describe old genome sequencing technique

A

create aligned library
sequence ordered fragments
read sequence in order dictated by clone map
- create genomic library and align and sequence in order and then can read sequence
too much - no one does this anymore

39
Q

describe new genome sequencing technique

A

create random library
sequence unordered fragments
align sequences clones by computer
requires deep sequence coverage >30x
see some sequences more than others so must sequence 30x genome to have complete coverage

40
Q

do all sequencing approaches need the PCR step

A

NOOOO
newer sequencing approaches skip PCR - single molecular sequencing

41
Q

describe single molecular sequencing

A

uses nanopore
strand is denatured
single stranded DNA will bind to motor protein
pulls DNA strand through motor protein
goes down to nanopore and electrical current
changes in current will depend on what base is there and sequence can be read - like a certain base changes sequence in a certain way

42
Q

name and describe advantages of nanopore - 3

A

sequencing single molecules opens the possibility of studying new biological questions - need to use very little DNA
very long reads minimize need for genome assembly and allows mapping of repetitive sequences
portable - very small, take anywhere like mac

43
Q

how does nanopore help map repetitive DNA sequences

A

lots of DNA is repetitive
hard to assemble since it does just repeat in a pattern - repeats in many places
comes in patches
easier to analyze and map genome assembly

BIOL 200 (29 decks)

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