Molecular Genetic Techniques 2 (L6)

Question 1

cDNA library screening goal

Answer 1

for obtaining a full-length coding sequence of the target gene for subsequent characterization

Question 2

cDNA library screening steps

Answer 2

1. cells from each colony on nitrocellulose filter
2. incubate filter w/ alkaline solution to lyse cells, denature DNA
3. hybridize w/ labeled probe
4. wash away labeled DNA that does not hybridize to DNA bound to filter
5. autoradiography
   signal appears over plasmid DNA that is complementary to probe

Question 3

plasmid

Answer 3

circular, extrachromosomal DNA that can replicate independently inside bacteria

Question 4

DNA cloning steps

Answer 4

1. Restriction enzyme - sticky ends on DNA vector
2. Restriction enzyme to cut plasmid.
3. Enzymatically insert DNA into plasmid.
4. Transform plasmid into bacteria.
5. Antibiotic resistance selection - only transformed cells survive.
6. Create large colony of bacteria w/ copies of DNA you want to amplify.

Question 5

what can be done to plasmids/what do they have that allows for DNA cloning?

Answer 5

1. polylinker region/ multiple cloning site where you can engineer a series of restriction enzymes sites
2. origin of replication - can replicate inside bacteria
3. ampicillin resistant gene

Question 6

reverse transcription mechanism

Answer 6

1. Hybridize mRNA w/ oligo-dT primer (recognize polyA tail)
2. Transcribe RNA into cDNA.
3. Remove RNA w/ alkaline conditions.
4. Add polyG tail at 3’ end of ss cDNA w/ terminal transferase
5. Hybridize w/ oligo-dC primer.
6. Synthesize complementary strand w/ DNA pol.
7. End w/ ds cDNA sequence

Question 7

how can you protect cDNA?

Answer 7

methylation at EcoR1 sites

Question 8

Once you have ds cDNA, what can you do with a plasmid?

Answer 8

Transform cDNA into a plasmid, then use same DNA cloning procedure to create more full length sequences

Question 9

how can a probe be synthesized from a plasmid?

Answer 9

add primer and nt’s to create a restriction enzyme site so that restriction enzyme sites are automatically incorporated into amplified DNA - then make probe by using DNA pol to transcribe ssDNA w/ sequence from target region

Question 10

fragile X syndrome diagnosis

Answer 10

done with southern blot

Question 11

FMR1

Answer 11

fragile x mental retardation 1 - gene that causes the disease

• get > 200 CGG or CpG island repeats in promoter
• when tri-nt are expanded, triggers methylation of CpG islands and of repeats -> gene repressed -> problems

Question 12

northern blot

Answer 12

used to compare mRNA expression in different conditions

-slight difference from southern blot: can also evaluate level of expression (thickness, darkness of band)

Question 13

what do microarrays tell you?

Answer 13

know which gene is expressing at what level in your sample

Question 14

genomics

Answer 14

study of whole genome of an organism

Question 15

microarray colors

Answer 15

green: decreased expression
red: increased expression

Question 16

heatmap

Answer 16

tells expression of genes in different patients - important concept: using gene profiles as a better means of studying diseases

Question 17

what is the technique for protein characterization?

Answer 17

SDS-PAGE = sodium dodecyl sulfate - polyacrylamide gel electrophoresis

Question 18

what is the purpose of SDS?

Answer 18

detergent - denatures protein and adds an even coating of negative charge on the polypeptide

Question 19

why do you need a reducing agent for protein characterization?

Answer 19

to break disulfide bonds to get single polypeptide chains coated with SDS

Question 20

what are two reducing agents?

Answer 20

mercaptoethanol and dithiothreitol

Question 21

ratio of SDS to aa’s and implications

Answer 21

1 SDS/ 2 aa’s - nice even coating

Question 22

SDS-PAGE steps

Answer 22

1. Denature with SDS.
2. Place mixture of proteins on gel and apply electric field (proteins separated by size).
3. Stain to visualize separated bands (Coomassie blue).

Question 23

western blot

Answer 23

1. SDS-PAGE
2. Transfer to membrane.
3. Incubate with primary antibody (binds target protein).
4. Wash.
5. Incubate with enzyme-linked secondary antibody (binds primary antibody)
6. Wash.
7. React w/ substrate for enzyme on secondary antibody.
8. Visualize.

Question 24

what is the purpose of using two rounds of antibodies in western blot?

Answer 24

signal amplification

Question 25

clinical application of western blot

Answer 25

research and diagnosis (HIV)

Question 26

protein separation w/ 2D gel

Answer 26

1. separate proteins by isoelectric focusing

2. separate proteins by size (SDS-PAGE)

Question 27

isoelectric focusing

Answer 27

create a pH gradient, then apply electric field -> proteins migrate to where they are neutral

Question 28

isoelectric point

Answer 28

point where polypeptide has no charge

Question 29

advantage of 2D gel separation?

Answer 29

gives finer map of protein distribution

Question 30

3 types of column chromatography

Answer 30

1. gel filtration
2. ion exchange
3. antibody affinity

Question 31

basic concept of column chromatography

Answer 31

use a polymer matrix to separate proteins based on different properties

Question 32

gel filtration chromatography

Answer 32

smaller proteins get stuck in beads, large ones cannot -> larger proteins move through column faster, come out first

Question 33

ion exchange chromatography

Answer 33

polymer has charge that will bind proteins of opposite charge - ones w/ same charge come out first, then elute other proteins bound to column

Question 34

antibody affinity chromatography

Answer 34

only target proteins bind to antibody on the beads of the column -> gives you a concentrated, purified fraction

Question 35

proteomics

Answer 35

large scale analysis of proteins

Question 36

mass spectroscopy

Answer 36

used to identify and sequence proteins