Probing Southern blots & plaque lifts

– purpose: identify specific DNA fragments/clones

– using hybridization to specific probe

1. prepare blot – done this week

2. prehybridization (1-2 hours)

– prevents non-specific binding of nucleic acid probe

– reduces background

– blots incubated in solution containing

– high salt concentration (promotes nucleic acid binding)

– blocking agents – bind to surface of blot

– fill all available binding sites on membrane

– no space left for non-specific probe binding

examples:

– Denhardt’s reagent

– mix of high molecular weight polymers

– expensive

– BLOTTO – powdered milk preparation

– cheap

– high RNAase levels

– problem on Northern blots

– salmon sperm DNA

– bovine serum albumen (BSA)

– formamide – optional

– reduces rate of nucleic acid hybridization

– less false positives from similar sequences

3. labelled probe (prepared during prehybridization)

– may be:

– double stranded DNA – must be denatured before use (99°C, 5 min.)

– cloned restriction fragment

– cDNA

– PCR product

– may be in plasmid vector – easy to prepare

– problem

– hybridization of vector with blotted DNA

– e.g. pUC vector with kb ladder markers

– may be RE digested out of vector & eluted from gel

– no vector hybridization

– may be labelled by:

– random primer extension (Lab 5)

– DNA denatured

– second strand synthesized

– mix of different 6-bp oligo primers

– 4 dNTPs (1 is alpha-³²P)

– Klenow fragment

(large fragment E. coli DNA polymerase I)

– has 5'-3' exonuclease removed

– polymerase extends primers

– using denatured DNA as template

– alpha-³²P dNTP incorporated in new strand

– produces high specific activity probes

– nick translation

– DNA nicked with DNAase I (single-stranded nicks) – nicked template used for DNA synthesis

– 4 dNTPs (1 is alpha-³²P)

– E. coli DNA polymerase I (holoenzyme)

– has 5'-3' exonuclease activity

– DNA synthesis primed by 3' nicked ends

– 5'-3' exonuclease removes 5' side of nick

– replaced by radiolabelled sequence

– added by 5'-3' polymerase

– nonradioactive probes

– may use modified nucleotides

– bind fluorescent dyes or enzyme substrates

– added to 3' end of DNA

– terminal deoxynucleotidyltransferase

(terminal transferase)

– adds non-specific dNTPs to 3' end of DNA

– or bind enzymes to denatured DNA

advantages: safer than radiolabelled dNTPs

problems: activity does not last as long as radiation

– harder to see faint signals

other probes:

– single stranded DNA (oligonucleotides)

– labelled with T4 polynucleotide kinase

– transfers gamma-³²P from gamma-³²P ATP to 5' ends of DNA or RNA

– RNA – requires clone in T7/T3 promoter vector

– phage RNA polymerase – 4 NTPs (1 is alpha-³²P)

4. hybridization (usually overnight)

– blot incubated in hybridization solution

– fresh prehyb solution + labelled probe

– hybridization conditions – depend on probe used

– high stringency

– many H-bonds required for stable hybridization

– results from: high temperature

lower salt concentration

formamide

advantages of high stringency – fewer false positives

problems

– short probes (oligos)

– may not be long enough to stay H-bonded

– probes with mismatches – e.g. from different species

– may not have enough homology

– if problems occur, require lower stringency

– produced by changing 3 factors above

5. wash – wash excess probe & hybridization solution off blot

– several washes, stringency depends on probe

6. expose blot to X-ray film

– length of time depends on strength of signal

– minutes to days (radioactive probes only)

equipment used for hybridization

– traditional/low-tech/cheap – “seal-a-meal” bags

– clear plastic bags

– can be melted shut with electrical apparatus

– used to hold blot & radioactive hybridization solution

– incubated with shaking (even spread of probe)

problems:

– bubbles inside bag

– may prevent even contact of probe with blot

– must be carefully excluded from bag

– sealed off separately

– wet plastic – will not seal properly

– leaks of radioactive solution

– should seal inside second bag

– or incubate in tupperware container

– newer/specialized/expensive solution

– hybridization ovens

– maintain constant temperature

– contain rollers for specialized bottles

– blot(s) placed in bottle

– hyb. solution added – incubated overnight