Techniques

Question 1

x-ray crystallography

Answer 1

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

Question 2

NMR

Answer 2

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

Question 3

Gel filtration chromatography

Answer 3

• 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

Question 4

Ion exchange chromatography

Answer 4

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

Question 5

Affinity chromatography

Answer 5

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

Question 6

Gel electrophoresis with SDS polyacrylamide gel

Answer 6

• 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)

Question 7

Edman Degradation

Answer 7

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

Question 8

Western blot

Answer 8

• 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

Question 9

Gel electrophoresis separates DNA based on _______

Answer 9

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

Question 10

Melting temperature of oligonucleotides determines the temperature at which __________ occurs

Answer 10

hybridization

Question 11

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

Answer 11

DNA fragments, complex, hybridization

Question 12

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

Answer 12

true

Question 13

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

Answer 13

Southern blot

Question 14

Southern blotting steps

Answer 14

• 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

Question 15

SDS-PAGE separates proteins based on _______

Answer 15

size

Question 16

Northern blotting is used to detect a specific _______

Answer 16

RNA sequence

Question 17

Western blotting is used to detect

Answer 17

proteins

Question 18

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

Answer 18

antisense

Question 19

Microarray analysis is used to _________

Answer 19

simultaneously analyze expression patterns of thousands of genes

Question 20

What does the fluorescence in a microarray tell you?

Answer 20

mRNA abundance with a bunch of different probes

Question 21

The key reagent in a Sanger sequence is ___________

Answer 21

dideoxynucleotides

Question 22

Sanger sequence

Answer 22

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

Question 23

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

Answer 23

complementary

Question 24

PCR

Answer 24

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

Question 25

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

Answer 25

doubles

Question 26

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

Answer 26

DNA template, DNA primer, deoxyribonucleotides

Question 27

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

Answer 27

southern blot

Question 28

Allele-specific PCR can be used to determine _________

Answer 28

genotyping of DNA polymorphisms

Question 29

DNA fingerprinting uses

Answer 29

variable number tandem repeats (repeated DNA sequencing)

Question 30

electrophoresis

Answer 30

- polarized electrical field in a porous gel | - smaller DNA strands move through faster than larger

Question 31

restriction fragment length polymorphism

Answer 31

- 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

Question 32

steps of DNA fingerprinting

Answer 32

- PCR - electrophoresis (detection of different size fragments) - compare samples against target

Question 33

restriction digestion analysis

Answer 33

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

Question 34

hybridization

Answer 34

- 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

Question 35

Primers are important for _______ and ______ techniques

Answer 35

DNA sequencing, PCR

Question 36

steps of paternity test

Answer 36

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

Question 37

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

Answer 37

MstII

Question 38

Steps in testing for sickle cell

Answer 38

1. digest patient's DNA with "diagnostic" restriction enzyme 2. southern or PCR analysis 3. electophoresis

Question 39

What do massively parallel functional assays assess the function of?

Answer 39

protein variants

Question 40

Steps for massively parallel functional assays

Answer 40

- 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