Cell Bio Exam 2 Ch12

Front 1

Colony lift hybridization

Back 1

How do we select the colony with the desired cDNA?

1. Place nitrocellulose filter on plate to pick up cells from each colony.

2. Incubate filter in alkaline solution to lyase cells and denature released plasmid DNA hybridize with labeled probes

3. Perform autoradiography.

4. Incubate with labeled DNA.

5. Wash away labeled DNA that does not hybridize to DNA bond to filter.

6. Perform autoradiography to find colony.

Front 2

Autoradiography

Back 2

Use 3H because of its very short emission path

Emission is a beta-particle (i.e. a high speed electron)

3H-thymidine to label DNA

3H-uridine to label RNA

3H-amino acid to label protein

For colony lift, use 32P-probe and x-ray film

Front 3

Shuttle vectors

Back 3

URA3+ is a selectable marker gene (analogous to ampr

Ura3- yeas cells require uracil

Front 4

Origins of DNA replication

Back 4

ARS for eukaryotes

ORI for prokaryotes

Front 5

Overlapping restriction fragments

Back 5

What's need to make a genomic library?

Front 6

Chromosome walking

Back 6

Why use only a partial digestion with very low concentrations of restriction enzyme?

Important to have the genomic library contain overlapping fragments for sequencing or linkage studies

Call ________________.

Ex: Sau3A and BamHI leave the same compatible sticky ends.

Front 7

Leland Hartwell

Back 7

Generated many cdc mutations in yeast

cdc mutations block cell cycle progression at various points

used genetic complementation to determine which mutations were alleles of each other

Front 8

Fundamental (Molecular) Complementation Screening

Back 8

1. A single cdc mutant is grown at permissable temp.

2. Transform yeast by treatment with LiOAC, PEG, and heat shock

3. Plate and incubate at permissive temperature on medium lacking uracil

4. Replica plate and incubate at non-permissive temperature

Only colonies carrying a wild-type CDC gene are able to grow

MUST KNOW

Front 9

Paul Nurse

Back 9

Started his career at Guinness brewery
Yeast geneticist and molecular-cell biologist

Re-isolated the "red" plasmid from the rescud yeast cells and sequenced the red gene

Deduced the amino acid sequence to identify the protein product
---these proteins control the cell cycle; his and other labs determined how these regulatory proteins work

Front 10

DNA Gel Electrophoresis

Back 10

Cut insert out of vector.

Resolve on agarose gel.

Large DNA fragments more more slowly than do smaller fragments.

Stain with ethidium bromide and it fluoresces in UV light

Good for .5-2 kbps

Polyacrylamide gels are used for 20-1500 bps

Front 11

Pulse-field electrophoresis

Back 11

Agarose again, but current turned off and on at right angles

Resolves huge DNA fragments:
--genomic fragements
--20 kbp - 10 mega bp

--cut the genome with "8-base pair cutters" v "6-bp"

Front 12

Physical map

Back 12

Restriction enzymes provide a _______________.

Front 13

Polymerase chain reaction (PCR)

Back 13

Probably the most powerful molecular biology technique ever devised (by Kerry Mullis)

Amplifies specific DNA sequences, but any that you choose
--Forward and reverse primers select sequence of DNA to amplify
Primer = oligonucleotide

Primers are in vast molar excess compared to original template DNA

Exponential increase (replication) of target DNA template

Front 14

Cycles in PCR

Back 14

1. Denaturation of DNA and annealing of primers

2. Elongation of primers using special heat-stable DNA polymerase

3. Termination

Can also design primers to include restriction sites

Front 15

DNA sequencing (3 techniques)

Back 15

1) Maxam and Gilbert
2) Sanger's dideoxy chain termination technique
3) next generation DNA sequencing

Front 16

Maxam and Gilbert techniqe

Back 16

End label, then chemically cleave the DNA
Not used much anymore

End label: 32P added to 5' ends by a kinase