Molecular Techniques Flashcards
How to Clone
- isolated wild type genomic DNA
- cut up DNA with restriction endonuclease
- clone DNA into a plasmid
DNA cut up by endonuclease
- 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.
resistance markers
- grow in media with antibitoic
- only cells with antibiotic resistance plasmid will grow
addiction modules
- cells grown in poison
- plasmid contain antidote gene
- poison is long lasting
- antidote is short lived
- must replicate plasmid to live
EcoRI type II
- 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.
Classes of restriction endonucleases
- Class I
- Class II
- Class III
Class I
- multisubunit enzyme that requires SAM, ATP, and Mg2+
- cut DNA at distant site
Class II
- only require Mg2+ and usually cut with palindromic recognition site
Class III
- requires ATP
- not commercially available
Class I mechanism
- 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)
Using Restriction enzymes in cloning
- over 300 available
- each one cuts at a unique DNA sequence
- most 4 to 6 base pair palindromic sequences
Steps to cloning with restriction enzymes
- extract DNA from bacteria
- cut DNA with restriction enzyme
- cut vector with same restriction enzyme
- incubate DNA fragments with vector and seal with ligase + ATP to seal phosphate backbone
- transform ligated products into E. Coli
- grow bacteria on plates containing antibiotic
(whatever resistance cloned for) - screen the colonies for inserted DNA
When DNA is cut by restriction enzyme
- it is staggered
- only staggered ends cut with same restriction enzyme will go back together
plasmid issue
- each colony that grows will carry a plasmid
- the problem is the plasmid has compatible ends with itself
- selection for those with correct insert.
Blue White/Screening
- 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
How to fish out interesting genes
- 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.
Typical Assays
- enzyme activity
- activity of a “reporter”
- mRNA concentration
- protein concentration (western Blot)
reporter gene
coding region for the gene
promoter activity
- 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.
Reporter gene protocol
- clone promotor for gene you are studying
- clone the coding region for the reporter gene downstream of promotor
- put your reporter construct into organism
- grow cultures under the conditions the should should be on or off
- assay reporter gene.
B-galactosidase as a reporter gene
- use calorimetric assay
calorimetric assay
- 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.
Regulation with lacZ
- 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.
GFP
- 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.
methods of direct mRNA measurement
- northern blot
- qrtPCR
northern blot
- 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
qRT-PCR
- 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
intensity threshold
- assigned
- above background but before log phase
cycle #
inversely proportional to amount of starting mRNA
quantification
- takes place in thermocycler that monitor flour
- computer graphs fluoro versus cycle number
Western Blot
- measures amount of protein expressed
- uses an antibody against the protein
advantage of western blot
sometimes the amount of protein expressed is not entirely dependent on the mRNA expressed
SDS-PAGE
- 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
primary antibody
antibody that recognizes your protein
secondary antibody
- 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.
