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Scientific Tools in Medicine

Blotting techniques for the study of DNA, RNA, and proteins

Peter C Hayes, C Roland Wolf, John D Hayes

Identifying and measuring specific proteins in complexbiological mixtures, such as blood, have long beenimportant goals in scientific and diagnostic practice.More recently the identification of abnormal genes ingenomic DNA has become increasingly important inclinical research and genetic counselling. Blottingtechniques are used to identify unique proteins ornucleic acid sequences. They have been- developed tobe highly specific and sensitive and have becomeimportant tools in both molecular biology and clinicalresearch.

General principleThe blotting methods are fairly simple and usually

consist of four separate steps: electrophoretic separa-tion of protein or of nucleic acid fragments inthe sample; transfer to and immobilisation on papersupport; binding of analytical probe to the targetmolecule on paper; and visualisation of bound probe.Molecules in a sample are first separated by electro-phoresis and then transferred on to an easily handledsupport medium, or membrane (table). This immobil-

Membrane supports and applicationis

Support medium Mitacromolecules bound

Nitrocellulose paper'', DNA, RNA, protcinDiazophenyl paper DNA, RNA, proteinCharge modified nylon sheets' ProteiniPolvvinylidine difluoride membrane' ProteinGlass fibre"':Aminopropyl ProteinQuaternarNy ammonium ProteinPhenvlenediisothiotcvanatc P'rotein

of interest. The synthesis of such probes is describedlater in this series. '4' Southern blotting is a DNA-DNA hybridisation technique that is often used toidentify, for example, the location of a single gene onchromosomal DNA (fig 1).

Before Southern blot analysis can be carried out

DNA

IEndonucleases

DNA fragments

Agarose gE

Blotting

Departments of Medicineand Clinical Chemistry,Royal Infirmary, Universityof Edinburgh, EdinburghEH3 9YWPeter C Hayes, MD, lecturer inmedicineJohn D Hayes, PHD, lecturerin clinical chemistry

Imperial Cancer ResearchFund MolecularPharmacology and DrugMetabolism Laboratory,Department ofBiochemistry, University ofEdinburgh, EdinburghEH8 9XDC Roland Wolf, PHD,laboratory head

This series has been editedby Drs P C Hayes andJ D Hayes, Royal Infirmary,Edinburgh.

BrAledj 1989;299:965-8

ises the protein or DNA fragments, provides a faithfulreplica of the original separation, and facilitates subse-quent biochemical analysis. After being transferred tothe support medium the immobilised protein or nucleicacid fragment is localised by the use of probes, such asantibodies or DNA, that specifically bind to themolecule of interest. Finally, the position of the probethat is bound to the immobilised target molecule isvisualised, usually by autoradiography.Three main blotting techniques have been de-

veloped and are commonly called Southern, northern,and western blotting. Southern blotting, named afterits originator,' allows DNA fragments to be identifiedwith probes of DNA, which hybridise by hydrogenbonding to complementary fragments of chromosomalDNA. Northern blotting" allows individual moleculesof messenger RNA (mRNA) to be identified andmeasured after hybridisation to their correspondingDNA sequences (the sequences of DNA from whichthe mRNA was transcribed). By contrast, westernblotting allows particular proteins to be identified withspecific antibodies used as analytical probes. 2

MethodsSOUTHERN AND NORTHERN BLOTTING

A prerequisite for both Southern and northernblotting is the availability of cloned DNA or syntheticDNA sequences that can be used as probes for the gene

Incubation withradiolabelled cloned

Position of DNAfragmentscomplementaryto probe

FIG 1 -Southern blotting

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fragments of DNA must be produced from chromo-somes by enzymic digestion with restriction endo-nucleases. These enzymes are obtained from micro-organisms and digest double stranded DNA at specificcleavage sites determined by the nucleic acid sequence.As restriction endonucleases'cleave DNA at only, thesecleavage sites each enzyme produces a characteristicnumber of fragments from a single specific length ofDNA. Allelic variants of a particular gene can bedetected if the differences in the nucleic acid sequenceinclude the cleavage sites because digestion by theappropriate restriction enzyme will yield differentfragments from allelic genes (fig 2). Hence mutationsmay result in either the loss of cleavage sites or theintroduction of new, additional cleavage sites. Suchdifferences in the fragments produced from one geneare referred to as restriction fragment length poly-morphisms (RFLPs).

After enzymic digestion of chromosomal DNA theresulting DNA fragments are subjected to electro-phoresis in agarose, which separates the fragmentsaccording to size, the smaller DNA fragments migrat-ing furthest in the electric field."' The electrophoresisgel is then covered with a sheet of nitrocellulose, on towhich the DNA is absorbed. Before hybridisation milddenaturation in alkaline solution ensures that theblotted DNA is single stranded. The nitrocellulosesheet is then incubated with the gene probe, which isusually cloned DNA.4 The gene probe will have beenradiolabelled, and it hybridises by hydrogen bondingto single stranded chromosomal DNA fragments thatcontain complementary nucleic acid sequences. Thenitrocellulose sheet is then dried and covered by aphotographic or x ray film to allow localisation of thegene under examination (fig 3).

Northern blotting entails essentially the same pro-cess described above except that complementary DNAis used to probe RNA. Messenger RNA is firstseparated according to size by electrophoresis and thentransferred to paper before gene probes are used tolocate the message of interest. With this methodthe transcripts of specific genes can be studied andmeasured.

(fI12 3

t4

t

Oil 2 3

1 y 2 3 4

1 x 2 3 4

FIG 2-Sites of enzyme digestion by endonucleases: (a) cleavage ofDNA at positions 1, 2, 3, and 4; (b) cleavage with loss of restrictionsite 2; (c) cleavage with additional restriction sitey; (d) cleavage withadditional restriction site x arising in region corresponding to gene probe(blackened region)

1 2 3 :4 .. 5 6, 1,7... 8. 9 10 i:T ;12 813 1415*_Af. ., " Q ;;' .~'!I'

-,;Qf 0 ' !''szr' ... . CO-s * # rLj ZA

FIG 3 Example ofSouthern blotting Fften rows of different DNAfragments separated by electrophoresis anid identified with cytochromeP-450 complementary DAA probe

WESTERN BLOTTING

The specific antibodies to the prdtein under exami-nation are needed for western blotting. The sourcesof these antibodies may be serum samples (forexample, from patients with known infections), puri-fied polyclonal antibody preparations, or monoclonalantibodies.'6As a consequence of the interaction of antibody and

antigen the target protein (the antigen) when immobi-lised on a paper support can be identified withantibodies. The mixture containing the protein underinvestigation is first resolved into its componentsby electrophoresis in polyacrylamide gels,'" and theproteins are then transferred to nitrocellulose paper(fig 4). The primary antibody binds to its target proteinand is immobilised on the' blotting support. Theantigen-antibody complex can then be identifiedwith antibodies to the primary antibodies, which arelabelled with radioactive iodine (fig 5), and can then bevisualised by autoradiography. The labelling antibodycan be removed and the protein studied further.

ApplicationsSOUTHERN BLOTTING

Less than 10 years ago DNA polymorphisms werefirst identified in' the II globin gene for haemoglobin.'7Since then DNA probes have been used increasingly inmedical research, in particular for identifying genesassociated with genetically transmitted diseases so thatrestriction fragment length polymorphisms have nowbeen found in Huntington's chorea,'9 familial growthhormone deficiency,'9 sickle cell anaemia,2" adultpolycystic kidney disease, and cystic fibrosis." Therecent application ofSouthern blotting to cystic fibrosishas incriminated a DNA sequence very close to anallele'named pJ3.11 which provides a crucial clue in thesearch for a "cystic fibrosis" gene. Once the structureof the gene is known it should not be long before theputative protein thought to be causing the disease isidentified.24 Investigation of diseases with less obviousgenetic transmission, such as ischaemic heart disease,have used DNA probes with provocative results.With gene probes for apolipoprotein B certain alleles



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were identified in many patients with ischaemic heartdisease. Interestingly, none of these alleles was asso-ciated with variations in cholesterol or apolipoproteinB concentrations, suggesting that genetic variation atthe locus for apolipoprotein may be an independent,though unknown, risk factor for ischaemic heartdisease.

Such investigations have therefore improved ourunderstanding of many diseases and have also greatlyimproved our ability to diagnose some of them beforesymptoms develop. Also, antenatal diagnosis has greatpotential in genetic counselling and is already beingapplied to such conditions as Duchenne type andBecker's muscular dystrophy.26

Southern blotting has also been applied to investiga-tion of malignant disease. For example, hepatocellularcarcinoma, one of the commonest cancers in the world,

Solution containingmixture of proteins

Polyacrylamide gel

0oj

Ec)o

Electrophoresis

Blotting

Filter paper

Nitrocellulose

Polyacrylamide

Incubation with radiolabelledantibody or antiserumand then radiolabelleda nti -anti body

.: t..4-

Position of protein(antigen) identifiedby antibody

FIG 4-Western blotting

FIG 5-Example of western blotting: (a) 10 samples separated byelectrophoresis and different proteins identified with a general proteinstain; (b) the specific polypeptide YTk picked out by antibody to Yk

has been recognised for years to be associated in manygeographic regions with hepatitis B virus infection.Blotting techniques have shown that DNA from thehepatitis B virus is incorporated into the genome ofboth hepatocellular carcinoma cell lines and tumours.7Such information increases our understanding ofcarcinogenesis and should lead to improvements intreatment and prevention.

ProblemsThe usefulness of these tests relies on gene linkage

analysis when the exact site of the abnormal gene isunknown, as often happens. The probes used usuallydetect nucleic acid sequences close to, rather thanwithin the affected gene. As "swapping" recombina-tion occurs between genes of paired chromosomes, inabout 5% of samples the probe marker will no longer bephysically associated with the abnormal gene, resultingin a probability of detecting carriage of the gene of only95%.7X The accuracy of this method obviously varies,depending on how close the nucleic acid sequence thatis being identified is to the gene under study: the largerthe distance the higher the probability of error. Such alimitation does not apply to probes for genes alreadyknown to be responsible for the disease.A further potential problem of applying these

techniques to inherited diseases is that of hetero-geneous genes for clinically similar disorders. Thusinformation relating to a patient's pedigree is essentialfor accurate analysis of certain diseases, such as

autosomal dominant manic depressive disease, inwhich genetic heterogeneity is known to exist.8

NORTHERN BLOTTING

Northern blotting, which uses DNA probes thathybridise with complementary RNA sequences, is anideal tool to study the products of gene transcription.For example, overexpression of certain proteins inneoplastic cells can be examined to determine whetherthe increase is due to gene amplification (an increase inthe number of genes), is regulated at a transcriptionallevel due to gene derepression (where one gene givesrise to increased mRNA), or is due to post-transcrip-tional mechanisms such as reduced cellular excretion.Gene amplification has been shown to be responsiblefor acquired drug resistance in tumour cells in smallcell carcinoma, where methotrexate resistance is con-

ferred by amplification of the gene for dihydrofolatereductase.9 Multiple drug resistance, conferred by the


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multi-drug resistance gene (MDR-1), has been shownin vitro to be associated with increased RNA intumours,'" although whether this is due to geneamplification or gene derepression is unknown.

WESTERN BLOTTING

Western blotting is currently widely used in clinicalresearch. Individual proteins from a protein mixturesuch-as plasma or other body fluids can be identifiedand then "rescued" and further studied by removingthe labelling antibody, making the technique a power-ful tool. It is gaining widespread application particu-larly for research into the acquired immunodeficiencysyndrome. The method allows serum samples frompatients to be screened for antibodies against a varietyof viral antigens. It uses the antibodies in the serum as"probes" to show the presence of known viral antigensthat have been run on an electrophoretic strip. By theuse of standard-that is, positive-antisera, antigensfrom different sources can be studied and cross reactiv-itv identified. In this way closely related HIV-I andHIV-II can be detected and differentiated."' Thespecificity of western blotting is also used to checkpositive results ofenzyme linked immunosorbent assay(ELISA) tests for the AIDS virus, thereby reducingthe risks of finding false positive results,"2 the-techniquehas also been applied to other viral diseases, includinghepatitis B. It can be used, for example, to test forantibody production in the serum ofpatients vaccinatedwith synthetic vaccines.

Western blotting has also proved invaluable inprotein chemistry applied to such areas as enzymologyand oncology-for example, to find different iso-enzymes and their pattern of distribution in diseasedand healthy tissue. Similarly, the identification andlocalisation of tumour markers within neoplasticmaterial can easily be studied with the appropriateantisera.

Future developmentsThe application of blotting techniques will un-

doubtedly increase in the next decade. It is likely thatrather than being employed in isolation they willcomplement other areas of technological advanceoutlined in this series. An example of this is thepolymerase chain reaction, in which special heatresistant DNA polymerase enzymes are used to dupli-cate very small segments ofDNA that would ot-herwisego undetected. Once amplified such DNA segmentsare identified by Southern blotting. This technique hasbeen used to identify infection with HIV many monthsbefore antibodies can be detected in the patient'sserum.34

Blotting techniques, which allow detection ofgenomic material or proteins fairly simply and cheaplywith high specificity, are widely used in medicalresearch. Nevertheless, the results depend on thepurity of the materials used and the technical skill ofthose using them.

We thank Dr J Miles for helpful criticism, Drs N Hastieand R Meehan for providing the examples of Southernblotting, and Mrs J Johnstone for typing the manuscript.

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2 Thomas P. Hybridization of denatured RNA and small DNA fragmentstransferred to nitrocellulose. Proc Natl Acad Sci USA l980;77:5201-5

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4 Alwine JC, Kemp D)J, IParker BA, et al. Detection of specific RNAs or specificfragments of DNA bv fractionation in gels and transfer to diazobenzsylox -

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methyl paper and detection with antisera: a method for studying antibodyspecificity and antigen structure. Proc NadltAcadSci USA 1979;76:3166-20.

Glossary

Genomic DNA - DNA derived fromchromosomes

Probe - an agent that will accuratelyidentify a specific nucleicacid or amino acidsequence

Autoradiography -the process whereby theradioactivity of isotopes isdetected by photographicfilm applied on to theisotope labelled probe

Hybridisation -the process of bonding of asequence of nucleic acid-bases to complementarybases

Specificity - the ability to identify asubstance accurately,allowing few false positives

Sensitivity - the ability to identify alltested substances, allowingfew false negatives

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22 White R, Woodward S, Leppert M, et al. A closely linked genetic mark-er forcvstic fibrosis. Nature 1985;318:382-4.

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polymorphisms associated with myocardial infarction. N Engl J M1ed1986;315: 1509-15.

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