Restriction endonucleases

-where derived from

-site of action

-result

bacteria-derived enzymes that cleave foreign DNA (but not bacteria's own DNA, which is protected by methylation)

cleave at site of inverted repeats 4-6 bp long

create breaks with sticky or blunt ends

Southern Blot

DNA digested w restriction endonuclease

frags separated on agarose gel, transferred to nitrocellulose fiver

DNA then can be labeled with probes, which hybridize w complementary DNA

PCR (polymerase chain reaction)

-function

-requirements

synthesizes large # of copies of DNA from a single copy

requires: spec double-stranded seq to be amplified, oligonucleotide primers (20-30, single-stranded), DNA polymerase, thermocycler (denaturation, annealing, primer extension)

Dot blot

molecules NOT separated by electrophoresis

target molecule deteccted with probes or antibodies

provides no info on size of molecule, only its presence/absence

QUICK

probes

-composition

-how they work

can be made of DNA or RNA

complimentary to DNA seq (100% match)-->remain bound, otherwise washed off

ASO (allele specific oligonucleotides)

short seq of synthetic DNA (15-21 bases) that are complementary to target DNA

probe used to detect a specific allele

must be labeled with radioactive, enzymatic or fluorescent tag

RFLP (restriction fragment length polymorphism)

if cleavage site contains a mutation, restriction endonucleases cannot cleave-->results in longer DNA fragments

VNTR (variable number tandem repeats)

short repeated DNA seq

number of repeats is inherited, but varies in diff individuals

can be used for parental identification or personal identification (CODIS)

genomic library

entire DNA of an organism

cDNA library

reverse transcriptase is used to synth cDNA from mRNA present in cells

cDNA library contains transcripts of expressed proteins of a particular cell type/tissue

no introns or other non-coding regions