Tools of Molecular Genetics Flipped Classroom Flashcards
cDNA
made as copy of RNA, no introns, used to determine AA sequence or make large quantities of protein by cloning
Dideoxyseq
Utilize polyermase and chain terminating nucs
READ FROM THE BOTTOM UP!!!! (longer goes further)
Microarray
large # of short DNA molecules that are immobilized
Each DNA fragment acts as probe for certain gene
Have two mRNA-whicheever lights up more has more expression of the gene
Hybridization
have 2 complementary nucleic acid strands to form a duplex-used in detecting specific nucleotide sequences
Transfection
Euk cells take up DNA from surroundings
Transformation
Prok cells take up DNA from surroundings
Gene knockdown
Downregulate expression using RNAi
Palindromic
Two fold sum-looks the same twice in 360 degree turn
DNA electrophoreresis
Runs from + to -
Smaller move faster
May have to denature RNA
Ethidium bromide/radioisotope stain
Hybridization conditions
Higher-lower probablty of hybridization
-use formamide (denature) or more heat, shorter probe
Lower-higher probability of hybridization
-Use less formaminde and less heat, longer probe
RFLP
detect by running gel electrophoresis
3 blotting techs
North-RNA, South-DNA, West-Protein
Steps of blotting
- Extract whatever from where and purify
- Place into gel and run
- Transfer to nitrocellulose or nylon paper
- Add radioactive probe
- Wash and xray
Allele Specific Oligos
Match nucleotide seq flanking substitution, typically use wt and mutant copies of gene as probe, see if have mutant or wt copy in cell
Keep in mind each cell has two copies of protein, heaterzygotes will always show up as positive
PCR steps
- heat to separate strands
- Cool with excess of primers
- incubate with DNA poly and 4 deoxy nucs
Allele specific PCR
want to create a lot of specific allele
Must have near perfect 3’ end binding
RT PCR steps
- isolate mRNA
- create cDNA with revtranscriptase
- Primers
Automated DNA seq
PCR makes fragments that are separated by capillary gel electrophoresis-read color as go down tube
Construct cDNA library steps (and how to tell which DNA has what gene)
- Cleave DNA with endonuclease
- Use ligase to put fragments into plasmids
- Transfect into proks
+palce paper on proks, take off paper and add probe, take off and see what lights up under autoradiogrpahy
Serial cloning steps
- endonuc a plasmid
- ligate dna
- repeat as desired
can engineer hybrid proteins with fluorescent marker to see what took-or use reporter gene
Make large amounts of protein
Place next to strong promoter
Epitope tagging
epitope for specific antibody is fused to target protein
- useful for monitoring protein expression/localization/topological dynamics/interations
- can use to do column separation
Knokcout and replacement
no gene active and only mutant gene active
