Genetics: Lab Methods Flashcards
DNA Cloning Mech
Cleave plasmid w/ restriction nuclease, insert DNA fragment to be cloned, insert recombinant DNA into bacteria, propagate bacteria, purify recombinant bacteria
FISH
Hybridize fluorescent probes to chromosome, wash, visualize 2 regions of complementary DNA per chromosome
Southern Blotting Characteristics/Uses
Labor/time-intensive, but can detect large repeats and methylation status so used to detect Fragile X syndrome
Southern Blot Method
Restrictions create different size fragments, run through electrophoresis, can see the size of DNA that binds to probe and thus # of repeats
Quantitative PCR
Real time PCR
Gold Standard for Mutation Scanning
DNA Sequencing
Difference b/w Mutation Screening and Scanning
Screening: search for a limited # of known muts in a gene
Scanning: scan a gene for all possible muts, can detect both known and unknown muts
Allele Specific Oligohybridization (ASO)
Mutation screening method using radiolabeled probe of wt and mut to see which a person has (or both)
DNA Sequencing Mech
Put DNA strand and RNA primer in mixture w/ plenty of dNTPs to elongate chain but also rare ddNTP (sequentially). It will stop when it hits the ddNTP and you can see how long that fragment is. After doing all 4 just read to see the order
Linkage Analysis (when used and 3 necessary conditions)
Used in rare situations when causative mut can’t be detected and genomic localization of gene is known, informative markers are available, and multiple members can be studied
10 Things to Ask About For Pedigree
Early deaths Miscarriages Intellectual disability/developmental ability Birth defects Consanguinity Genetic disease/early onset disease Ancestry Country of origin Religion Last Names
2 Signs of Autosomal Recessive from Pedigree
Usually seen in single generation of family
Mothers and fathers equally likely to transmit/inherit
Signs of XLD
Only females present with it bc usually lethal in males
Sign of Incomplete Generation
“Skips” a generation
