DNA Techniques- Lecture 8/19/21 Flashcards
Restriction Endonuclease
Cleaves within the DNA at a particular sequence (palindromic sites)
Gel electrophoresis
When current is applied DNA runs to the positive side, bigger molecules move faster than small
Pulsed field gel electrophoresis
Used to separate big pieces of DNA, including chromosomes
Southern blot
Cleave DNA with restriction endonuclease->run on gel->transfer to nitrocellulose->Hybridize with labeled probe for gene of interest
Detecting trinucleotide expansion with southern blot
Larger the fragment, more trinucleotide expansions
Detecting point mutation using southern blot
If mutation is in cleavage site, will lead to abnormal fragment lengths
RFLP
Restriction fragment length polymorphism- when there is some polymorphism in the human genome that adds or takes away a cleavage site
FISH
Fluorescent in situ hybridization-> denature chromosomes and at fluorescent probe
Northern blots
Similar to southern blots, but with RNA
Western blots
Look for protein-> uses labeled antibodies to see if protein of interest is present
DNA band shifts
When a protein is interacting with a piece of DNA, it becomes larger and moves slower through the gel
DNA footprinting
When looking for a specific binding site, add DNase which nicks the phosphodiester bond, where there is a “shield” there is a protein binding site
Thermocycler
Machine used in PCR to heat up and cool off DNA to denature and re-anneal DNA
Primers
Short ~20 bop oligonucleotides that bind in both forward and reverse direction to the gene of interest to amplify fragments
Detecting expansion through PCR
PCR not viable for genes ~5,000 by and over because the polymerase falls off the DNA, will not get results
RT-PCR
Rat stands for reverse transcriptase, used to look for RNA in the sample by turning it into cDNA via reverse transcriptase
Oncotype score
Using RT-PCR, used to detect cancer genes in patients to give them a recurrence score, about 16 genes of interest
Molecular cloning
Cleave gene and plasmid with restriction enzyme, gene incorporates into plasmid via sticky ends, kept in library by bacteria
Sticky ends
EcoR1 makes “sticky ends” by creating complimentary base pairings because of the palindromic sequence
genomic library
Constructed directly from fragments of genomic DNA
CDNA library
Derived from mRNA, converted to DNA by reverse transcriptase first
Dideoxy analogue
Uses a dideoxy sugar which lacks 3’ hydroxyl to terminate chain elongation
Sanger method
Uses dideoxy sugars to randomly stop at a given location, know the opposite basepair is the opposite
