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M2M Unit 1 > Techniques > Flashcards

Techniques Flashcards

1
Q

x-ray crystallography

A
  • purify protein
  • crystallize protein (rate limiting step)
  • collect diffraction data
  • calculate electron density and fit residues into density
  • no size limit
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2
Q

NMR

A
  • purify protein (must be concentrated, relatively small!)
  • dissolve protein
  • collect NMR
  • calculate structure
  • don’t have to crystallize protein
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3
Q

Gel filtration chromatography

A
  • columns have beads
  • apply protein mixture on top of column, run solution through beads
  • big protein can’t get in pores (travel through columns quickly)
  • small proteins get trapped (travel slowly)
  • separate proteins based on size
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4
Q

Ion exchange chromatography

A

-cation exchange column is negatively charged, binds positively charged proteins

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5
Q

Affinity chromatography

A

protein (ie: glucose-binding protein) attaches to (glucose) residues on beads and everything else washes through

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6
Q

Gel electrophoresis with SDS polyacrylamide gel

A
  • proteins are denatured
  • SDS is negatively charged and proteins are coated in negative charge
  • under electric field, small proteins move faster than large ones (because they have less negative charge)
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7
Q

Edman Degradation

A
  • N-terminal end labeled with chemical, becomes labile
  • break bond, get labeled first AA
  • gets less accurate as you go
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8
Q

Western blot

A
  • run protein sample on gel
  • transfer onto membrane
  • incubate with primary antibody (antibody specific to protein of interest)
  • antibody binds to membrane, wash away other antibodies
  • add secondary antibody and color
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9
Q

Gel electrophoresis separates DNA based on _______

A

size (molecules move through pores in gel at rate inversely proportional to chain length)

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10
Q

Melting temperature of oligonucleotides determines the temperature at which __________ occurs

A

hybridization

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11
Q

Southern blotting allows detection of specific ___________ from a ________ mixture via ________

A

DNA fragments, complex, hybridization

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12
Q

T/F: For gel electrophoresis, all of the DNA must be identical

A

true

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13
Q

If you have a complex mixture but want to identify a specific DNA sequence, use ________

A

Southern blot

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14
Q

Southern blotting steps

A
  • cleave DNA with restriction enzymes
  • electrophoresis on a gel
  • denature DNA duplex
  • transfer to nitrocellulose paper (alkaline solution denatures DNA)
  • add probe that is complementary to DNA/RNA you want
  • reduce temp. to just below Tm for specific hybridization
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15
Q

SDS-PAGE separates proteins based on _______

A

size

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16
Q

Northern blotting is used to detect a specific _______

A

RNA sequence

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17
Q

Western blotting is used to detect

A

proteins

18
Q

In a northern blot, the probe has to be _________ in order to see the band

A

antisense

19
Q

Microarray analysis is used to _________

A

simultaneously analyze expression patterns of thousands of genes

20
Q

What does the fluorescence in a microarray tell you?

A

mRNA abundance with a bunch of different probes

21
Q

The key reagent in a Sanger sequence is ___________

A

dideoxynucleotides

22
Q

Sanger sequence

A

Have all four dNTPs and add one of four ddNTPs; ddNTP stops sequence, can read sequence

23
Q

In a Sanger sequence, the end product DNA sequence is (identical/complementary) to the starting sequence

A

complementary

24
Q

PCR

A
  • denature strands (95C)
  • primer sits in site (anneals) (55C)
  • DNA pol extends primers (72C)
25
Q

In PCR, the number of DNA molecules _____ with each cycle

A

doubles

26
Q

In PCR, the substrates include ________, __________, and _________

A

DNA template, DNA primer, deoxyribonucleotides

27
Q

Use _________ to detect restriction fragment length polymorphisms (sickle cell)

A

southern blot

28
Q

Allele-specific PCR can be used to determine _________

A

genotyping of DNA polymorphisms

29
Q

DNA fingerprinting uses

A

variable number tandem repeats (repeated DNA sequencing)

30
Q

electrophoresis

A
  • polarized electrical field in a porous gel

- smaller DNA strands move through faster than larger

31
Q

restriction fragment length polymorphism

A
  • digest patient’s DNA with restriction enzyme (MstII)
  • electrophorese against normal gene
  • southern blot with P32-labeled B-globin gene (mutated in sickle cell)
  • see size of fragments compared to normal
32
Q

steps of DNA fingerprinting

A
  • PCR
  • electrophoresis (detection of different size fragments)
  • compare samples against target
33
Q

restriction digestion analysis

A
  • quickest, cheapest

- DNA digested with enzymes that would indicate by sizes of DNA produced if DNA segment of interest had been inserted

34
Q

hybridization

A
  • used when screening many clones
  • add radioactive “probe” or complementary fragment and allow DNA to hybridize followed by exposure to x-ray
  • identify clone containing recombinant DNA with correct insert
35
Q

Primers are important for _______ and ______ techniques

A

DNA sequencing, PCR

36
Q

steps of paternity test

A
  1. PCR or restriction digestion of VNTRs
  2. southern blot
  3. electrophoresis
37
Q

Sickle cell anemia destroys a restriction site for the restriction endonuclease ______

A

MstII

38
Q

Steps in testing for sickle cell

A
  1. digest patient’s DNA with “diagnostic” restriction enzyme
  2. southern or PCR analysis
  3. electophoresis
39
Q

What do massively parallel functional assays assess the function of?

A

protein variants

40
Q

Steps for massively parallel functional assays

A
  • generate a library with mutations in a sequence of interest
  • multiplexed functional assay (measure activity of each variant before and after to see if variants are gain or loss of function mutation)
  • analyze changes in abundance to measure relative effect size

M2M Unit 1 (4 decks)

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