Restriction endonuclease
are in bacteria and attack foreign DNA to destroy it
restriction endonuclease have a
specific nucleotide sequence target site to cut
Eco-RI
endonuclease
palindromic
the strands are complementary and the endonuclease cut at specific places one each strand that are the same
purpose of sticky ends
cut in a way that allow the DNA to stick back together again
DNA has a ____ charge
negative
large molecules in gel electrophoresis go
not as far as smaller molecules
acrylamide is used in Gel electrophoresis when
separation needs to be at the nucleotide level
pulsed field gel electrophoresis is for
larger bits like chromosomes
blotting paper in Southern blot utilizes
capillary action
Southern blot is for
DNA
restriction maps
visualize different restrictive endonuclease sites
With Trinucleotide repeats a southern blot will yield a
larger piece on the blot
Sickle Cell Anemia
DNA mutation, point mutation, all individuals have the same mutation (no allelic homonegativity)
With sickle cell you get one larger marker on the Southern Blot because....
a diseased copy of the gene, and the endonuclease site isn't there
DNA fingerprinting
Several locations on a chromosome contain short tandem repeat regions that are targeted with sequence specific primers and have different numbers of tandem repeats resulting in different fragments
SNPs are analyzed using
DNA fingerprinting
RFLP
restriction fragment length polymorphism- the absence of a restriction cleavage site in one gene allele compared to another leads to a distinct restriction fragment lengths
Single stranded conformation polymorphism
used when a mutation changes the structure of single stranded DNA as measured by gel electrophoresis
FISH
Fluorescence in situ hybridization
FISH purpose
looks like a southern blot but used to check chromosome number or section
Northern Blot
same as southern but for RNA
Western Blot
similar to Southern but for proteins
Western blot differences
uses antibody as a probe , coated with SDS because proteins do not have a uniform charge
Mut S can be used to find
mismatched DNA in a southern blot
DNA foot printing
helps us to identify where a protein binds
Tac polymerase
used for PCR and found in hot springs, does not have clamps
PCR cannot amplify long strands of DNA because
tac does not have a clamp
dNTPS
needed for the extension of annealed primers by a heat stable DNA polymerase in PCR
SSCP
takes PCR a step further by denaturing the PCR product into single strands of DNA
RT-PCR
allows one to use PCR to amplify RNA
Molecular Cloning
`clone genes into plasmids , take the foreign DNA and chops with endonuclease. to create sticky ends
Eco-RI
endonuclease involved in moleculer cloning , like AATT
Ligase attacks
Eco-RI to plasmid
use of antibiotic resistance in a plasmid
isolates cells with the desired DNA
genomic library
constructed directly from fragments of genomic DNA
cDNA library
derived from mRNA , the mRNA is covered to DNA by reverse transcriptase and the cloned
Sanger sequencing
DNA has fluorescent markers on it and is elongated and terminated at each marker
Next generation sequencing
group of automated techniques used for rapid DNA sequencing