Molecular Techniques

Question 1

How to Clone

Answer 1

• isolated wild type genomic DNA
• cut up DNA with restriction endonuclease
• clone DNA into a plasmid

Question 2

DNA cut up by endonuclease

Answer 2

• foreign DNA (unmethylated) is cut up by restriction modification system - endonuclease and methylase
• will target the hemimethylated or unmethylated DNA because all DNA in the cell will be methylated.

Question 3

resistance markers

Answer 3

• grow in media with antibitoic

- only cells with antibiotic resistance plasmid will grow

Question 4

addiction modules

Answer 4

• cells grown in poison
• plasmid contain antidote gene
• poison is long lasting
• antidote is short lived
• must replicate plasmid to live

Question 5

EcoRI type II

Answer 5

• methylase - methyl group added to the 2nd adenine residue
• if methylated, EcoRI endonuclease will not cut it
• unmethylated sequence will be quickly cut up by EcoRI endonuclease.

Question 6

Classes of restriction endonucleases

Answer 6

• Class I
• Class II
• Class III

Question 7

Class I

Answer 7

• multisubunit enzyme that requires SAM, ATP, and Mg2+

- cut DNA at distant site

Question 8

Class II

Answer 8

• only require Mg2+ and usually cut with palindromic recognition site

Question 9

Class III

Answer 9

• requires ATP

- not commercially available

Question 10

Class I mechanism

Answer 10

• enzyme binds to recognition site
• DNA translocated through enzyme and cut at the distant site
• methylate and nuclease are part of the same complex (why they require SAM)

Question 11

Using Restriction enzymes in cloning

Answer 11

• over 300 available
• each one cuts at a unique DNA sequence
• most 4 to 6 base pair palindromic sequences

Question 12

Steps to cloning with restriction enzymes

Answer 12

1. extract DNA from bacteria
2. cut DNA with restriction enzyme
3. cut vector with same restriction enzyme
4. incubate DNA fragments with vector and seal with ligase + ATP to seal phosphate backbone
5. transform ligated products into E. Coli
6. grow bacteria on plates containing antibiotic
   (whatever resistance cloned for)
7. screen the colonies for inserted DNA

Question 13

When DNA is cut by restriction enzyme

Answer 13

• it is staggered

- only staggered ends cut with same restriction enzyme will go back together

Question 14

plasmid issue

Answer 14

• each colony that grows will carry a plasmid
• the problem is the plasmid has compatible ends with itself
• selection for those with correct insert.

Question 15

Blue White/Screening

Answer 15

• Uses the lacZ gene product (B-galactosidase) to determine if a piece of DNA has been cloned into the multiple cloning site of a vector.
• the multiple cloning site to cut plasmid to insert DNA is in middle of LacZ gene
• plasmids that have this insert do not make an inactive B-galactosidase enzyme
• plasmids that do not can still produce the enzyme
• put X-gal on plates, cells with empty plasmid will be blue.
• colonies that have plasmids containing the inserted will be white

Question 16

How to fish out interesting genes

Answer 16

• sequence all inserts
• DNA hybridization if you know the sequence
• screen for insert activity
• if you know insert activity, insert that into mutant and screen for it.

Question 17

Typical Assays

Answer 17

• enzyme activity
• activity of a “reporter”
• mRNA concentration
• protein concentration (western Blot)

Question 18

reporter gene

Answer 18

coding region for the gene

Question 19

promoter activity

Answer 19

• reporter fusions

- if studying the regulation of a protein that is not easy to assay, replace it with a protein that is easy to assay.

Question 20

Reporter gene protocol

Answer 20

1. clone promotor for gene you are studying
2. clone the coding region for the reporter gene downstream of promotor
3. put your reporter construct into organism
4. grow cultures under the conditions the should should be on or off
5. assay reporter gene.

Question 21

B-galactosidase as a reporter gene

Answer 21

• use calorimetric assay

Question 22

calorimetric assay

Answer 22

• the products of the assay produce a color
• use Xgal to determine if active in plates (blue/white)
• use ONPG which turns yellow when cleaved by B-galactosidase
• quantify using a spectrophotometer.

Question 23

Regulation with lacZ

Answer 23

• w/o trp - activation of trpEDCBA genes to make Trp
• w/ trp - repression of trpEDCBA genes
• TrpE is an anthranilate synthase (hard to measure
• replace with LacZ
• grow cells +/- tryptophan
• Add ITPG or X-gal
• measure amount of product with a spectrophotometer.

Question 24

GFP

Answer 24

• isolted in the jellyfish Aequora victoria which glows green
• small and folds easily so it is active in most bacteria
• fuse the coding region to the promotor you are studying and then look for fluorescence.
• since the substrate is light, you can view in live cells.

Question 25

methods of direct mRNA measurement

Answer 25

- northern blot | - qrtPCR

Question 26

northern blot

Answer 26

- RNA subjected to electrophoresis through agarose gel - separate all polymers based on size - label a DNA sequence, and use it to probe an RNA blot - RNA/DNA hybrid forms - band appears darker when more mRNA is present. - does not tell you absolute quantity

Question 27

qRT-PCR

Answer 27

- make DNA copy of RNA - bind specific DNA oligonucleotide primer complementary to 3' end of mRNA and extend with RTase - after DNA made add a second primer to further DNA amplificaiton - takes place in presence of sybr-GRN - fluoresces when bound to dsDNA

Question 28

intensity threshold

Answer 28

- assigned | - above background but before log phase

Question 29

cycle #

Answer 29

inversely proportional to amount of starting mRNA

Question 30

quantification

Answer 30

- takes place in thermocycler that monitor flour | - computer graphs fluoro versus cycle number

Question 31

Western Blot

Answer 31

- measures amount of protein expressed | - uses an antibody against the protein

Question 32

advantage of western blot

Answer 32

sometimes the amount of protein expressed is not entirely dependent on the mRNA expressed

Question 33

SDS-PAGE

Answer 33

- separates cellular proteins - each protein denatured and given a negative charge with detergent - given a uniform negative charge/mass ratio - loaded on top of gel and electrical field applied - all proteins migrate toward + electrode, the smaller proteins move fastest

Question 34

primary antibody

Answer 34

antibody that recognizes your protein

Question 35

secondary antibody

Answer 35

- the antibody that is used in the blotting - usually radioactive - visualize with this one - directed against antibody constant region - same for all antibodies in a species.