Week 4 - DNA and RNA analysis techiniques Flashcards
(21 cards)
methods for studying DNA
gel electrophoresis
restriction enzyme digest
southern blotting
DNA cloning
gel electrophoresis
Size of DNA (in case of linear DNAs), shape ( supercoil, linear, nicked, relaxed)
to know the size, linearize DNA/RNA first
use agarose (polysaccharide;horizontal) or polyacrylamide (soft plastic;vertical) due to negative phosphate backbone, move toward positive
intercalation
insertion of a compound between the bases
ethidium
4 rings; hydrophobic
intercalates; can damage DNA
fluoresces in UV
visualize DNA in a gel
restriction endonucleases
sequence specific DNA binding proteins
restriction enzymes bind DNA as dimers
recognize palindromic DNA sequences; two half-sites
Cleave DNA symmetrically on both strands of DNA
cutting results; restriction endonuclease; restriction enzyme results
blunt
sticky
frequency of digestion
(1/4)^n
ex. if recognized sequence is ATTCG, frequency of digetion on average is (1/4)^5
Restriction modification system
protect the host genome from foreign DNA
EcoR1 methylase - put CH3 where restriction enzyme will not cleave
Plasmid
circular extrachromosomal DNA molecule in bacteria
can be replicated independently from chromosomes
Cloning DNA
to make more DNA of one’s interest
Plasmid vector
To clone DNA
requires:
1. replication origin; to start the replication of plasmid
2. selectable marker gene; to sort out transformed cells
3. a cloning site; restriction sites where endonuclease can provide an insertion site
DNA ligase
Linearized plasmid and gene of interest can be recombined by this enzyme
recombinant plasmid
a plasmid that has gene inserted and rejoined by DNA ligase
DNA blot hybridization (South Blot)
detect the presence of a specific DNA sequence; method used to detect your wanted DNA sequence
- DNA applied to gel -> electrophoresis -> migration
- Salt solution -> Sponge -> Gel-> Nitrocelluose filter-> Papertowels; Salt solution ‘blots’ DNA onto filter by cappilary action
- DNA transffered to filter
- (filter in “seal-a-meal” bag) Hybridize with unique nucleic acid probe
- remove unbound probe and expose X-ray film to filter (autoradiodiagram)
Nucleic Acid probe
used in South blot which will bind to interested gene sequence (hybridizes with complementary DNA sequence)
End-labeling a DNA molecule
- Remove phosphates: 5’ ends are phosphate ends -> treat with phosphatase
- add 32P: 5’ ends will have -OH group -> treat with kinase plus gamma-32P-ATP
- Optional. for a probe with a single labeled end, remove one end with a restriction enzyme; the cut end will have regular P instead of 32P
methods for studying gene expression (mRNA)
RNA blot hybridization (Northern Blot)
RNA blot hybridization (Northern Blot) - very similar to Southern blot
detects the presence of a specific RNA sequence - okay success rate
- Separate RNA by size: extract RNA from sample, run electrophoresis
- transfer RNA to memberane
- use labeled probes to hybridize membrane
- visualize RNA on X-ray
Trouble in analyzing RNA
RNA is unstable
RNAase which digest RNA is found EVERYWHERE
To solve the problems in analyzing RNA; synthesis of cDNA
cDNA is a complementary DNA from mRNA
mRNA is unique = have a poly A tail
1. use poly A tail to isolate mRNA from other forms of RNA
2. use oligo dT to attach to mRNA poly-A-tail
3. have RT and dNTPs synthesize 1st strand of cDNA
4. remove RNA
5. cDNA (2 strand) will be made by random hexamers and DNA polymerase and dNTPs
- Now, the sequence is much easier to study
Microarray analysis
Northern blot; instead of membrane filter, you print.
Detects whole genome expression profile
*also involves hybridization
(Process) inserts from DNA clones + hybridized sample 1 and sample 2 => on microscope slide with array-> when put in computer analysis, (will either glow or not glow)
