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
what is in tubes for sanger sequencing
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
how many tubes used in sanger sequencing
4
27
name the chain terminators
ddATP (1mM) - red ddGTP (1mM) - purple ddTTP (1mM) - blue ddCTP (1mM) - green super low concentration compared to dNTPs
28
describe ddNTP chain terminators
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
describe sanger sequencing - gen
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
what can you see if sanger sequence ran on gel
bands corresponding to sequences can show all possible lengths (4 reactions) read sequences off gel
31
what are the limitations of sanger sequencing
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
what is next generation sequencing (NGS)
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
describe NGS technology
ligating the same linkers to a mixture of DNA fragments
34
describe NGS - first part
DNA denatured and anneals to complementary primers anchored on solid support PCR conducted (amplifies in a fixed spatial arrangement
35
describe NGS - second part
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
describe an image of NGs
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
describe assembly of whole genome sequences
getting a bunch of 100nts of info but want to know how they fit together
38
describe old genome sequencing technique
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
describe new genome sequencing technique
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
do all sequencing approaches need the PCR step
NOOOO newer sequencing approaches skip PCR - single molecular sequencing
41
describe single molecular sequencing
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
name and describe advantages of nanopore - 3
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
how does nanopore help map repetitive DNA sequences
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