SouthernBlot-

8/2/2019 SouthernBlot-

1/6

Southern Blotting andRelated DNA DetectionTechniquesTerence A Brown, University of Manchester Institute of Science and Technology, Manchester, UK

Southern blotting is a technique for transfer of DNA molecules from an electrophoresis gel

to a nitrocellulose or nylon membrane, and is carried out prior to detection of specific

molecules by hybridization probing.

Introduction

Southern blotting is one of the central techniques inmolecular biology. First devised by E. M. Southern (1975),Southern blotting results in transfer of DNA molecules,

usually restriction fragments, from an electrophoresis gelto a nitrocellulose or nylon sheet (referred to as amembrane), in such a way that the DNA banding patternpresent in the gel is reproduced on the membrane. Duringtransfer or as a result of subsequent treatment, the DNAbecomes immobilized on the membrane and can be used asa substrate for hybridization analysis with labelled DNAor RNA probes that specifically target individual restric-tion fragments in the blotted DNA. In essence, Southernblotting is therefore a method for detection of a specificrestriction fragment against a background of many otherrestriction fragments (Brown, 1999). The restricted DNAmight be a plasmid or bacteriophage clone, Southern

blotting being used to confirm the identity of a clonedfragment or to identify an interesting subfragment fromwithin the cloned DNA, or it might be genomic DNA, inwhich case Southern blotting is a prelude to techniquessuch as restriction fragment length polymorphism (RFLP)analysis.

In this article the general principles of Southern blottingare described, followed by overviews of the methodologiesused for preparation of DNA prior to blotting, for blottingitself, and for hybridization analysis. The article concludeswith a survey of the applications and limitations ofSouthern blotting.

The Principle of the Southern Blot

The ability of nitrocellulose powder or sheets to bind DNAhas been known for many years and was utilized in th

1950s and 1960s in various types of nucleic acid hybridization studies. In these early techniques the immobilizeDNA was unfractionated, simply consisting of total DNAthat was bound to nitrocellulose powder or spotted onto nitrocellulose sheet. The introduction in the early 1970s ogel electrophoresis methods that enable restriction fragments of DNA to be separated on the basis of their sizprompted the development of techniques for the transfer oseparated fragments en masse from gel to nitrocellulossupport. The procedure described by Southern (1975involving capillary transfer of DNA from the gel to nitrocellulose sheet placed on top of it, was simple aneffective, and although embellished over the years th

original procedure differs only slightly from the routinmethod still used in many molecular biology laboratories

The original methodology for Southernblotting

The original methodology for Southern blotting illustrated in Figure 1. An agarose electrophoresis gecontaining the fractionated restriction fragments, is placeon a filter paper wick that forms a connection between thgel and a reservoir of high-salt buffer. The nitrocellulos

Article Contents

Secondary article

. Introduction

. The Principle of the Southern Blot

. Preparation of DNA Prior to Southern Blotting

. DNA Blotting Techniques

. DNA/DNA Hybridization

. Types of Hybridization Probes

. Applications and Limitations of Southern Blotting

. Summary

Agarose gel

1 2 3

DNAmarkers

RestrictedDNA

Buffer

Wick

Support

Gel

Paper towels

Nylon membrane

Nylon membrane

Figure 1 Southern blotting. Reproduced from Brown (1999) with permission of BIOS Scientific Publishers Ltd, Oxford.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net


2/6


3/6

be treated with appropriate cell wall-degrading enzymes,and the same is true for plant cells though the latter areusually broken open by physical grinding of tissues thathave been frozen in liquid nitrogen. This causes somefragmentation of the DNA but provides a much higheryield.

After cell disruption, genomic DNA extraction proce-

dures continue with steps aimed at removing the majorbiochemicals other than DNA present in the initial extract.A protease such as proteinase K might be included in thebuffer used for cell disruption, in order to begin thedegradation of proteins in the extract, but deproteinizationis routinely carried out by phenol extraction, the additionof phenol or a 1 : 1 mixture of phenol and chloroformresulting in precipitation of proteins. After centrifugation,the precipitated proteins migrate to the interface betweenthe organic and aqueous phases, whereas the nucleic acidsremain in the aqueous phase. For plant extracts, whichcontain greater quantities of carbohydrates than are foundin most animal and microbial cells, an additional purifica-

tion involving the detergent cetyltrimethylammoniumbromide (CTAB) is frequently used. This compoundspecifically binds nucleic acids, improving their recoveryduring phenol extraction. Most extraction procedures alsoinclude digestion of RNA with ribonuclease and a finaltreatment with ethanol, which precipitates the remainingnucleic acid polymers, enabling ribonucleotides and otherlow-molecular weight contaminants to be removed. Theprecipitated DNA is dried and then redissolved in water ora TrisEDTA buffer.

Preparation of plasmid or bacteriophage DNA

Special techniques must be used if the objective is to obtainplasmid or bacteriophage DNA from bacterial clones.Plasmid DNA can be obtained by any one of severaltechniques that exploit the physical differences betweenplasmids and bacterial genomic DNA, plasmids beingsmall supercoiled molecules whereas genomic DNA, aftercell disruption, is present as long, linear fragments. Apopular method involves treatment of the cell extract withalkali, which precipitates the linear DNA but leavessupercoiled plasmids in solution. Bacteriophage DNA,for example recombinant l or M13 vectors, is usuallyobtained directly from bacteriophage particles secreted byinfected bacteria, the purification process being simplified

by the fact that these particles consist solely of DNA andprotein.

DNA Blotting Techniques

After the purified DNA has been treated with one or morerestriction endonucleases it is fractionated by agarose gelelectrophoresis and the gel then pretreated prior to setting

up the Southern blot. The pretreatment has two objectiveFirst, it is desirable to break the DNA molecules iindividual bands within the gel into smaller fragmentsbecause smaller fragments transfer more quickly thalarger ones. This is achieved by soaking the gel i0.25 mol L21 HCl for 30 min, which results in a smaamount of depurination cleavage of the b-N-glycosidi

bond between purine bases (adenine or guanine) and thsugar component of their nucleotides which is followeby decomposition of the sugar structure and breakage othe polynucleotide chain. The second pretreatment is witan alkaline solution that denatures the double-strandeDNA molecules by breakage of their hydrogen bonds, sthe molecules become single-stranded. This aids thetransfer andsubsequent binding to the membrane,and alsensures that after binding the base-pairing components othe polynucleotides are available for hybridization with thprobe.

If a nitrocellulose membrane is being used thenthe alkapretreatment is followed by neutralization of the gel b

soaking in a Tris-salt buffer, this step being essentiabecause DNA does not bind to nitrocellulose at a pH ogreater than 9.0. The Southern blot is then set up, aillustrated in Figure 1, with a high-salt transfer buffeusually the formulation called 20 SSC, whiccomprises 3.0 mol L2 1 NaCl (salt) and 0.3 molL2

sodium citrate. The same buffer can be used for transfeto a nylon membrane, but with a positively charged nylomembrane an alkaline transfer buffer (0.4 molL2 1NaOHis used because, as described earlier, this results iimmediate covalent attachment of the transferred DNAto the membrane. With this type of transfer the alkapretreatment is unnecessary. Theblot is thenleft for at leas

18 h for a high-salt transfer, or 2 h for an alkaline blot.After blotting, the transfer setup is dismantled and th

membrane rinsed in 2 SSC and left todry. If the blot habeen made onto a nitrocellulose or uncharged nylomembrane, then the DNA is only loosely bound to thmembrane at this stage. More permanent immobilizatiomust therefore be carried out, either by baking at 808C fo2 h, which results in noncovalent but semipermanenattachment of DNA to a nitrocellulose membrane, oUV irradiation, which results in covalent attachment oDNA to a nylon membrane.

DNA/DNA Hybridization

Hybridization analysis is based on the principle that twpolynucleotides will form a stable hybrid by base-pairingtheir nucleotide sequences are wholly or partly complementary. A specific restriction fragment in a Southern blocan therefore be detected if the membrane is probed with second, labelled DNA molecule that has the same, osimilar, sequence as the fragment being sought. We wi

Southern Blotting and Related DNA Detection Techniques



4/6

examine the types of hybridization probe that can be usedlater; first we will survey the methodology used forhybridization analysis.

Prehybridization of a Southern blot

Hybridization analysis is carried out by soaking theSouthern blot in a buffer containing the hybridizationprobe, usually in a tube that is constantly rotated so allparts of the membrane are exposed to the probe, oralternatively in a sealed plastic bag that is placed on ashaker.Hybridization is carriedout in two stages.First, themembrane is prehybridized in a solution designed to blockthe unused DNA binding sites on the membrane surface. Ifthis step is omitted then the probe will bind nonspecificallyto the surface of the membrane and the signal resultingfrom hybridization to the specific restriction fragment willbe difficult if not impossible to identify. The prehybridiza-tion solution therefore contains nonbiological polymeric

compounds such as polyvinylpyrrolidone and/or biologi-cal polymers such as Ficoll (a carbohydrate-basedcompound), bovine serum albumin or dried milk. DNAfrom an organism unrelated to the one whose DNA hasbeen blotted can also be used (salmon sperm DNA is apopular choice). Prehybridization takes between 15 minand 3 h at 688C, depending on the type of membrane.

The hybridization and washing steps

The second stage is the actual hybridization, which iscarried out in a high-salt buffer containing a detergent,

usually 2 SSC1 1% SDS. Two issues are critical at thisstage of the experiment. First, enough probe DNA musthybridize to the target restriction fragment to produce aclear signal that can be discerned by the detection systemappropriate for thelabel carried by theprobe. For the mostdemanding applicationsuch as detection of a single copygene in human genomic DNA, achieving sufficientsensitivity might be a problem even if the maximumamount of genomic DNA is loaded on to the electrophor-esis gel (in practice, about 10 mg of DNA per lane) and theprobe has been labelled to its maximal specific activity(4 109 dpmmg2 1 for a 32P radioactive label). Increasinglygreater problems will be encountered if a radioactive label

other than 32P is used, such as 35S, which is generallysuitable only for probing less complex DNAs such asrestricted plasmid or bacteriophage clones, or if anonradioactive probe is employed. Under these circum-stances some increase in sensitivity can be obtained byincluding an inert polymer such as 10% dextran sulfate or8% polyethylene glycol 6000 in the hybridization solution.These polymers are thought to induce the probe moleculesto form networks so that greater amounts of DNAhybridize to the target sites on the membrane. In practice,

a 10- to 100-fold increase in sensitivity can be achieve(Amasino, 1986).

The second critical factor that must be considered durinthehybridization stepis the specificityof the reaction. If thprobe DNA has been carefully chosen then it will containregion that is completely complementary to all or a part othe blotted restriction fragment that is being sought. If th

hybridizing region in the probe is not completely complementary to the target,then it willat least have a region ostrong similarity so that a stable hybrid can form. Thproblem is that the probe also has the potential to hybridizto any other blotted DNA fragments with which it hapartial complementarity. The hybridization experimenwill never give an unambiguous signal if the probe is morsimilar to a second restriction fragment than it is to the onbeing sought, but problems with nonspecific hybridizatiocan still arise even if the best match is with the specifitarget. This means that the hybridization step must bcarried out at a stringency that results in the specifiprobetarget hybrid remaining stable while all othe

hybrids are unstable, the stringency being determined bthe composition of the hybridization buffer and thtemperature at which the experiment is carried out. Buffecomposition is relevant because hybrid stability is dependent on the ionic strength and the presence of destabilizinagents, such as formamide, which disrupt hydrogen bondTemperature is relevant because the melting temperatur(Tm, the highest temperature at which the hybrid is stableof a fully base-paired hybrid is higher than that for one iwhich some base pairs have not formed because the proband target DNAs are not fully complementary. Formatioof the desired hybrid, and destabilization of nonspecifihybrids, can therefore be achieved by utilizing an appro

priate combination of buffer composition and hybridization temperature. A number of different strategies arpossible. If the probe is 4 100 bp in length, for example cloned restriction fragment, then the initial hybridizationusually carried out at 688C in a high-salt buffer, threpresenting highly stringent conditions under which onlstable hybrids are expected to form, with very little if annonspecific hybridization. With this strategy, washinsteps that are carried out after hybridization are designesimply to remove the nonhybridized probe. However, if short oligonucleotide probe is used (1525 nucleotides ilength), then the hybridization step is usually carried ouunder conditions of low stringency (typically at

temperature several degrees below the calculated Tm fothe desired hybrid), so that all potential hybrids, includinnonspecific ones, are able to form. Specificity is theachieved by a series of washesat increasing temperatures sthat, hopefully, only the desired hybrid remains at the enof the procedure.

After washing, the membrane is subjected to thdetection procedure appropriate for the label that habeen used, for example autoradiography for a radioactivlabel. Subsequent reprobing is possible if the membrane i


4 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net


5/6

stripped by washing in a high-temperature buffer contain-ing alkaliand detergent to destabilize the hybridized DNA.This procedure is never wholly satisfactory as it is difficultto avoid removing some of the blotted DNA at the sametime, so even nylon membranes carrying covalently boundDNA can only be reprobed ten or so times.

Types of Hybridization Probes

Any DNA molecule that is complementary to therestriction fragment being sought can be used as the probe.Depending on the application (see below), the probe mightitself be a restriction fragment, or it might be a DNAmolecule obtained by the polymerase chain reaction(PCR), or a cDNA prepared by complementary DNAsynthesis from an mRNA template. Synthetic oligonucleo-tides are also frequently used as hybridization probes,especially when discrimination between very similar target

DNAs is required. This is because oligonucleotides of 1525 nucleotides in length form hybrids whose meltingtemperatures can be estimated from the formula:

Tm5 (2 number of A and T nucleotides)

1 (4 number of G and C nucleotides) 8C

The Tm values for most 1525mer oligonucleotides fallbetween 40 and 908C. A single nonbase-paired nucleotideposition in the hybrid between the oligonucleotide probeand target DNA can reduce the Tm by 358C, so carefulcontrol of the posthybridization wash temperature candistinguish a target site that is completely complementary

to the probe from one that differs at just a single nucleotideposition, enabling highly specific probing to be carried out.

A second popular type of hybridization probe is onemade of RNA rather than DNA. RNA probes have anumber of advantages compared with their DNA counter-parts, the most important of which in practical terms is theavailability of methods for synthesis in the testtube of largeamounts of an RNA probe from a DNA molecule that hasbeen cloned adjacent to a promoter for a bacteriophageRNA polymerase. Starting with 1 mg of cloned DNA,purified T7 RNA polymerase,for example, can make up to30 mg of RNA in 30 min, this RNA being labelled with aspecific activity of over 109 dpmmg2 1 if a 32P-labelled

ribonucleotide is included in the reaction mixture. RNAprobes also have the advantage of being single-stranded,which means that the effective probe concentration doesnot decline during the hybridization, as happens with adouble-stranded DNA probe due to base-pairing of thetwo complementary probe molecules to one another.

Applications and Limitations ofSouthern Blotting

The applications of Southern blotting are diverse and arnot easily summarized in a short article. Two examples wisuffice to illustrate the range of research questions to whic

the technique can be applied.

Southern blotting in clone identification

The commonest applications of Southern blotting occuduring projects aimed at identification and cloning of specified gene. Southern blotting of genomic DNA is useto identify one or more restriction fragments that contaithe gene being sought and, after cloning and tentatividentification of the desired recombinant by colony oplaque hybridization probing, Southern blotting of restricted clone DNA is used to confirm the clone identifica

tion and possibly to locate a shorter restriction fragmencontaining the sequence of interest, from within the cloneDNA. The latter application is important because thinteresting gene sequence might be just a few kb in lengtbut contained initially in a genomic clone of severahundred kb prepared with a high-capacity vector such as bacterial or yeast artificial chromosome. For Southerblotting to be applicable to geneidentification it is of coursnecessary to have a suitable hybridization probe, one thawill detect specifically the one or more restrictiofragments that contain the gene being sought. Often thgene will have already been cloned as a cDNA that can bused as a probe for identification of the genomic version o

the gene. Alternatively, if the gene has been cloned from related organism, one in which the gene sequence is likelto be similar to that in the organism now being studiedthen heterologous probing can be used, in which somnoncomplementarity between probe and target is tolerateduring the hybridization step. This approach can be usedfor example, to identify homologues of human genes in thgenomes of other primates, and can even be used acrosbroad species barriers, for example by using Drosophilgenes as probes for related human sequences. Anothepossibility is to use the amino acid sequence of the proteicoded by thedesired gene to designa mixed oligonucleotidprobe, one that consists of a variety of related sequence

that together include all the combinations that would codfor a short segment of the amino acid sequence. Foexample, the mixed oligonucleotide 5-AA(C/U)GA(AG)AUGUU(C/U)UGGUA(C/U)GG-3, which containsixteen different sequences, covers all possibilities for thamino acid sequence asparagine-glutamine-methioninephenylalanine-tryptophan-tyrosine-glycine and would bexpected to detect the DNA sequence coding for thprotein segment when used at a sufficiently high stringencin a Southern hybridization experiment.




6/6

Restriction fragment length polymorphismanalysis

The second major application of Southern hybridization isin the technique called restriction fragment length poly-morphism (RFLP) mapping, which is important inconstruction of genome maps (Brown, 1999). Treatment

of genomic DNA from different individuals with a singlerestriction enzyme does not always give the same set offragments because some restriction sites are polymorphic,being present in some individuals but absent in others,usually because a point change in the nucleotide sequencechanges the restriction site into a sequence not recognizedby the restriction enzyme. These polymorphic sites can beused as markers in genetic mapping and were of consider-able importance in construction of the first comprehensivehuman maps (Donis-Keller et al., 1987), there being about105 RFLPs in the human genome. RFLPs are typed bySouthern blotting as shown in Figure 2, the probe being aDNA fragment that spans the polymorphic region

(possibly one of the polymorphic fragments, possibly afragment prepared by cutting the DNA with a differentrestriction enzyme, or possibly a fragment obtained byPCR) and the presence or absence of the polymorphic sitedetermined from the size(s) of the restriction fragment(s)detected after Southern blotting.

Summary

Southern blotting is a technique that enables a specificrestriction fragment to be detected against a background of

many other restriction fragments. It involves transfer ofDNA fragments from an electrophoresis gel to a nitro-cellulose or nylon membrane in such a way that the DNAbanding pattern present in the gel is reproduced on themembrane. Hybridization probing is then used to detectthe restriction fragment that is being sought. The basicmethodology for Southern blotting has not changed sincethe original technique was described in 1975, but modifica-tions have been introduced with the aim of speeding up theprocess and achieving a more efficient transfer. Southernblotting has many applications in molecular biology,including the identification of one or more restrictionfragments that contain a gene or other DNA sequence of

interest, and in the detection of RFLPs used in construction of genomic maps.

References

Amasino RM (1986) Acceleration of nucleic acid hybridization rate b

polyethylene glycol. Analytical Biochemistry 152: 304307.

Bittner M, Kupferer P and Morris CF (1980) Electrophoretic transfer

proteins and nucleic acids from slab gels to diazobenzyloxymeth

cellulose or nitrocellulose sheets. Analytical Biochemistry 102: 459

471.

Brown TA (1999) Genomes. Oxford: BIOS Scientific Publishers.

Donis-Keller H, Green P, Helms C et al. (1987) A genetic map of th

human genome. Cell51: 319337.

Southern EM (1975) Detection of specific sequences among DN

fragments separated by gel electrophoresis. Journal of Molecul

Biology 98: 503517.

Further ReadingAusubel FM, Brent R, Kingston REetal. (eds) (1993) Current Protoco

in Molecular Biology. New York: John Wiley.

Brown TA (1995) Gene Cloning: An Introduction, 3rd edn. Londo

Chapman & Hall.

Brown TA (1998) Molecular Biology Labfax. London: Academic Pres

Dyson NJ (1991) Immobilization of nucleic acids and hybridizatio

analysis. In: Brown TA (ed.) Essential Molecular Biology: A Practic

Approach, vol. 2, pp. 111156. Oxford: Oxford University Press.

Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning.

Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harb

Laboratory.

Watson JD, Gilman M, Witkowski J and Zoller M (1992) Recombina

DNA, 2nd edn. New York: WH Freeman.

Nylon membrane Autoradiograph

Hybridizing

bonds

2 3

R1 R2 R3

DNA probe

Restrictionsite map

Polymorphic site

Figure2 Detectionof an RFLP by Southern blotting.In this example, lan2 contains DNA that lacks the polymorphic restriction site R2, and socontains a single restriction fragment that hybridizes with the probe. TheDNA in lane 2 contains the polymorphic site and so yields two fragment

that hybridize with the probe. Reproduced from Brown (1999) withpermission of BIOS Scientific Publishers Ltd, Oxford.


6 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Documents

SouthernBlot-