southern blot and hybridization

southern blot

finds DNA using DNA

edward southern

studies gene orientation RS mapping

detect hologous genes across species

mutations deletions ++

diagnosis of cancer

southern blot componenets

genomic DNA, RE, agarose gel

nitrocellulose or nylon membrane used

probe oligonucleotide to find dna interested

detection radiation chemiluminescent or colormetric

not enough dna for ethid bromide

methodology

10ug DNA fragged

electropho .7-1.2% agarose gel

dsdna denatured to allow transfer

gel soaked in alkaline solution containing sodium hydroxide

neutralized in Tris solution

Pre transfer steps

depurinate DNA with HCL

denute dna with 0.5N NaOH/1.5MNaCl

neutralize DNA with Tris HCL 0.5M pH 7.2/1.5M NaCl for nitrocellulose alkaline buffer rocommened for nylon membrane

steps

dna isolated and cut with RE

frags separated by gel elec

transfer to nitrocellulose or nylon membrane

probe with oligonucleotide or PCR

detection

Norther blot analysis

northern looks at gene expression RNA is not digested prior to elec

run denat on gel to suppress 2ndary structures allows proper sizing

uses DNA to find RNA

Nitrocellulose vs Nylon

nitro hydrophobic binding nylon covalent

nitro fragile nylon durable

>200 bp <200 bp

lower bg higher bg

cannot be exposed to basic, can

not easily reprobed, can be reprobed many times

transfer methodology

transffered through capillary action

long time

large dna frag require depurination prior to denaturation

different transfer methods

vacuum and electrophoretic

southern blott app

genetics, oncology, typing class org, cloning verification of cloned dna, forensics.

principals of NA hybridization

ionic strength, higher salt harder to separate

base composition

AT less stable than GC in aq solution w NaCl

more GC BP harder to separate DNA higher Tm

decrease NaCl strands split easier

decrease hybrid temp more mismatched seq

inc %gc content stronger dna dna int

increase length of probe/primers stronger dna dna int

western blot

looks for protein using antibodies

george stark

mod of southern

separates proteins with PAGE

Fixing DNA on membranes

must adhered stably

nitrocellulose 80C 2 hr

Nylon expose to low level UV at 254nm

positively charged nylon