Applications to study respiratory disease: methods biology

Thorax 1990;45:147-153

Molecular biology and respiratory disease 2 Series editor: R A Stockley

Applications to the study and treatment of

Applications to the study and t'reatment ofrespiratory disease: methods in molecular biology

C A Owen, R A Stockley

In the first article in this series we covered basicgene structure and function. The aim of thissecond introductory article is to provide anoutline of the techniques commonly used inmolecular biology and to provide examples ofthe application of these techniques to theinvestigation and diagnosis of respiratorydisease.

with "blunt" ends it is often necessary tolengthen them in an asymmetrical manner inorder to produce artificial "sticky" ends tofacilitate annealing. Because of theirspecificity, restriction enzymes are alsoimportant in the detection of restrictionfragment polymorphisms (see below).

Restriction endonucleasesRestriction endonucleases are bacterialenzymes that cleave specific base sequences ofdouble stranded DNA. They are bacterial inorigin and digest foreign DNA to prevent itsincorporation into the genome of the bac-terium. More than 350 restriction enzymeshave been isolated, and between them theyrecognise some 85 distinct DNA sequences.They are named by three letter abbreviationsderived from the bacterial species from whichthey have been isolated; for example, Eco R I isone of the enzymes derived from the R strain ofEscherichia coli.There are two classes of restriction enzymes.

Class 1 enzymes recognise a specific nucleotidepair sequence and then cleave the DNA at anon-specific site a fixed distance away from thisrecognition site. Class 2 enzymes cleave theDNA only at the recognition site and always ina specific manner. Thus class 2 enzymes aremore important in recombinant DNA techno-logy because the cleavage is predictable. Class 2enzymes are referred to as "sequencespecific"-that is, they recognise only specificsequences, one for each enzyme. Thesequences are usually three to six base pairs inlength and possess a twofold rotationalsymmetry (palindrome). The number of cutsthat each enzyme makes in a particular DNAmolecule depends on the number of times theparticular recognition sequence is present inthe DNA. Some enzymes cut both strands ofDNA between adjacent nucleotide pairs toform so called blunt ends but most makestaggered cuts in the sequence to produce stickyends (see table). This latter effect is useful injoining one fragment of DNA to another frag-ment derived from a different source whenboth have been cleaved by the same enzyme(for example, in the construction of cloningvehicles). The overlapping complementarysequences at the ends of the fragments will joinspontaneously by hydrogen bonding (anneal).Indeed, when DNA fragments are produced

Nucleic acid hybridisation and DNAprobesDouble stranded DNA can be dissociated(denatured) by heating or by treatment with analkaline solution. If the temperature is sub-sequently lowered or the solution neutralisedthe two strands will reassociate (anneal). Thisprocess is highly specific and will occur onlybetween complementary strands. If two DNAsequences from different sources are denaturedand allowed to reassociate, base pairing willtake place only between any complementarysequences to form a duplex (hybridisation).

Probes are fragments of single stranded DNAor RNA that have been replicated in bacteria(cloned) to yield millions of identical copies.These fragments are labelled and used toidentify (probe) complementary sequences in amixture of nucleic acids. If a complementarysequence is present the probe will hybridisewith the target sequence and the resultingduplex will be identified by the label on theprobe. Probing may be carried out with nucleicacid that has been chemically extracted fromtissues, digested into fragments by restrictionSequence specificities and sites of cleavage of somecommonly used restriction endonucleases

Recognitionsequence with

Enzyme position of cut Product

BamH I 5'-GGATCC-3' -G GATCC-3'-CCTAGG-5' -CCTAG+ G-

Sticky ends

Eco R I 5'-GAATC-3' -G + AATC-3'-CTTAG-5' -CTTA G-

Sticky ends

Hae III 5'-GGCC-3' 5'-GG CC-3'3'-CCGG-5' 3'-CC + GG-5'

Blunt ends

Sma I 5'-CCCGGG-3' 5'-CCC + GGG-3'3'-GGGCCC-5' 3'-GGG CCC-5'

* Blunt ends

LungImmunobiochemicalResearch Laboratory,General Hospital,BirminghamCA OwenR A StockleyAddress for reprint requests:Dr R A Stockley,Clinical Teaching Block,General Hospital,Birmingham B4 6NH.

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enzymes, separated by gel electrophoresis, andblotted on to nitrocellulose. Alternatively, theprobe can be used directly on histologicalsections or whole chromosomes (in situhybridisation).There are two basic requirements for

hybridisation with a probe-namely, that thetarget sequences are accessible and that bothare in the form of a single strand.DNA probes can be constructed in three

ways:1 chemical synthesis alone to obtain apredetermined nucleotide sequence (forexample, synthetic oligonucleotide probes-seebelow);2 cleavage of genomic DNA and subsequentidentification and amplification ofthe fragmentof interest in an appropriate vector;3 treatment of messenger RNA (mRNA)with reverse transcriptase to producecomplementary DNA (cDNA). This is the mostcommonly used method and is usually appliedto mRNA obtained from tissues producing theprotein of interest. As a result fewer DNAspecies have to be screened than when thewhole genome is used. The cDNA probesconsist only of the coding sequences (exons)and no introns are included.DNA probes are used to determine the

chromosomal locations of genes, analysegene structure, diagnose single gene defectsby the detection of restriction fragmentpolymorphisms, and determine the tissuespecificity and activity of gene transcription(see below).

Reverse transcriptionReverse transcriptase is an enzyme that wasoriginally isolated from a group of viruseswhose genome consists of RNA rather thanDNA. The enzyme converts the viral genomeinto DNA (a process termed reverse transcrip-tion), which enables it to be inserted within thehost cell DNA and hence lead to viral replica-tion by the cell.The enzyme is most commonly used to

synthesise complementary DNA probes frommRNA. ThemRNA ofinterest is purified froma tissue in which it is being actively translated(for example, reticulocytes are used to obtainhaemoglobin mRNA). The microsomal frac-tion of the tissue is separated and the totalcellular RNA is purified and passed down acolumn containing either poly-T (thymine) orpoly-U (uracil) residues linked to a solid phasesupport. These residues bind the poly-A(adenine) containing (coding) mRNA whileother types ofRNA pass through. The mRNAis subsequently eluted from the column andreverse transcriptase is used to synthesise acomplementary DNA (cDNA) on the singlestranded mRNA template. An oligonucleotidecomplementary to the poly-A tail (oligo dT) isused as a "primer" DNA molecule. Ithybridises with the poly-A tail and is extendedby reverse transcriptase to produce a DNAcopy of the mRNA. The RNA is then des-troyed by treatment with alkali and the singlestranded cDNA is again copied to"give a doublestranded DNA molecule with the help of the

enzyme DNA polymerase. A final enzyme,called Sl nuclease, is then added to remove theremaining short sequence of single strandedDNA, producing a matched double strandedcDNA molecule for subsequent cloning.

CloningCloning refers to the insertion of fragments ofDNA into bacteria to produce multipleidentical copies (clones). DNA is not taken upefficiently in its native state by bacteria. Thusthe fragments are inserted into specialisedforms of DNA (vectors) that can enter andreplicate within the bacteria. The choice ofvector usually depends on the size of the DNAfragment to be cloned: plasmids are 0-10kilobases, bacterial viruses or bacteriophages5-20 kilobases, and cosmids are hybrid vectorscomposed of plasmid and bacteriophage DNAsequences (40-50 kilobases).

Plasmids are used preferentially for the clon-ing of gene probes when possible. They are selfreplicating units of circular double strandedDNA, which have a single cleavage site for oneof the bacterial restriction enzymes and usuallycarry genes encoding antibiotic resistance. Forexample, the synthetic plasmid pBR322 carriesresistance genes for both ampicillin andtetracycline and within the latter gene is thesingle restriction site for the enzyme Bam H1(fig 1). Incubation of the plasmid with thisenzyme will result in cleavage of the plasmid atthis site alone, leaving "sticky" ends. If theDNA to be cloned (insert) is also digested withBam HI similar "sticky end" sequences areproduced. When the two forms of DNA aremixed some of the sticky ends of the plasmidwill associate, by hydrogen bonding, with theDNA to be inserted. The two fragments can bejoined covalently by the enzymeDNA ligase toform a new "recombinant" DNA plasmidmolecule. The recombinants are thenintroduced into bacterial cells for propagationby a process called transformation. The fewbacteria which have been transformed by therecombinant molecules are identified by thepresence and type of antibiotic resistance thatthe plasmid has introduced (fig 1). The trans-formed bacteria are then grown in bulk cultureand lysed and the plasmidDNA is harvested bycentrifugation. The restriction enzyme used tocreate the recombinant molecule is then used toextract the insert, which is separated from theplasmid DNA by gel electrophoresis. In thisway a large quantity of identical copies of theoriginal DNA fragment may be obtained.

Probing and blottingSOUTHERN BLOTTINGSouthern blotting, devised byEM Southern, isa procedure in which a radiolabelled DNAprobe is used to identify a specific sequence ofDNA in a complex mixture ofDNA fragmentsthat have been transferred on to a nitrocellulosemembrane.'

In brief, genomic DNA is extracted fromblood leucocytes or other tissues and digestedby various restriction enzymes to produce

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Molecular biology methods

, Bam HI restriction siteTetracycline resistance

i gene

Cleave withBam Hi

Ampicillinresistancegene

pBR 322 Mixfra

den

cl1OR

Insert withintetracycline

gene(inactivated)

Recombinantmolecule

Incubate withbacteria

A No plasmid

BQ

Sensitivetetracycli

Repaired plasmid Resistanltetracycli

C Recombinant plasmid ResistaniSensitive

G of appropriate restriction sites. The fragmentsGwill be identified on the Southern blot and their

CCTAG c sizes are determined by reference to molecularG C weight markers run simultaneously on the

electrophoresis gel. If this process is repeatedwith several restriction enzymes, alone or invarious combinations, a restriction map of thegene can be constructed and the molecularbasis for certain genetic diseases may become

with DNA apparent.igments Deletion of a gene will result in the abolitionived from of any hybridisation signal. Partial gene3am Hi deletion or single base mutations may result ineavage the loss or creation of restriction sites for a

restriction enzyme and therefore lead to aTetracycline change in the size pattern of fragmentsene intact generated and identified on the Southern blot.

For example, in sickle cell anaemia a single basesubstitution (GAG -. GTG) results in the lossof a recognition site for the enzyme Mst 11 .

The abnormal gene can therefore be detectedby digesting DNA with Mst 11, which resultsin the production of larger fragments than

Repaired normal. These can be detected by Southernmolecule blot hybridisation with a fB globinDNA probe.

Southern blotting has also been performedwith oligonucleotide probes, which can detecteven single point mutations in intact DNA

to ampicillin and sequences. Oligonucleotides complementary toine both the normal and the mutant forms of the

gene are synthesised. These oligonucleotidesare radiolabelled and used to probe Southernblots of DNA prepared from samples from

ittompiilinnnormal subjects or those who are eitherine heterozygous or homozygous for the DNA

defect. Under appropriate conditions theprobes will hybridise with their complemen-tary sequences and distinguish the normal andmutant alleles. This technique has been used to

It to ampicillin detect the point mutation of the Z variant of theto tetracycline a, antitrypsin gene.3

Figure 1 Cloning of afragment ofDNA by construction of a recombinant plasmid andtransformation by competent bacteria. Selection of recombinant plasmids is made byexploiting the antibiotic resistance genes.

multiple DNA fragments of varying length,which are separated according to size by gelelectrophoresis (fig 2). The gel is soaked inalkali to denature the double stranded DNAfragments and it is then transferred to anoverlying nitrocellulose membrane. The mem-brane thus acquires an imprint of the separatedsingle stranded DNA fragments. The DNA isfixed on to the membrane by baking andexposed to a solution containing the radio-labelled single stranded probe, which bindsthrough hybridisation to any complementaryDNA sequences. The membrane is washed toremove unbound probe and the signal is de-veloped by autoradiography to show the posi-tions of the fragments bearing sequences com-plementary to that of the probe that has beenused.

Southern blotting can be used to analysegene structure by the construction of restrictionmaps. When genomic DNA is digested with arestriction enzyme it will cleave the gene ofinterest into several fragments, whose numberand nature depend on the presence and position

Restiendon'

dige

_.E- DNA fragmenlblotted onto

nitrocellulosemembrane

rctionuclease DNAstion fragments

Long /fragments

0ts owts ~ Agarose

- gel

I 0(incubated with Shortradiolabelled single fragmentsstfanded probecomplementary togene X +autoradiography

- Position of gene X

AutoradiographyFigure 2 The Southern blot technique.

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RESTRICTION FRAGMENT LENGTHPOLYMORPHISMSThe human genome is highly polymorphic(variable between individuals). On average, 1in 250 of the bases is polymorphic and 1 in 6 ofthese polymorphisms results in the loss or gainof a recognition site for one of the restrictionenzymes. Most of the polymorphisms aresituated in the 95% of the genome that is non-coding and therefore do not affect the pheno-type of the individual. The polymorphisms arerevealed as changes in the length of DNAfragments produced by the restriction enzymeson Southern blotting when an appropriateDNA probe is used. To be clinically useful,however, the polymorphism must be rare in thehealthy population and common in the diseasestate. Thus if the restriction fragment lengthpolymorphism is closely and stably linked tothe abnormal gene (linkage disequilibrium) itcan be used as a genetic marker for the diseaseeven if the true gene defect is not known. Forexample, in the United States among the blackpopulation the sickle cell mutant is associatedin 60% of cases with a polymorphism close tothe gene, which eliminates a site for the enzymeHpa I,' though the use of this marker is inferiorto analysis with Mst 11 (see above).

Unfortunately the use of restriction frag-ment length polymorphism is limited becausefew diseases are the result of single mutations,and even these may not alter restriction sites.

LOD scoresWhen diseases are not known to be linked tospecific genes the search for genetic linkagemay be tedious and time consuming. Theprocess depends on the identification of a genepolymorphism that is inherited together withthe disease. This means the application ofrestriction fragment length polymorphism tolarge families where the disease can beidentified and where several generations areavailable for study. The process is tedious asthe gene is unknown and hence all genes mayhave to be studied. Furthermore, becausethe specific defect is unknown a linkedpolymorphism has to be found and this couldoccur with any one of the 350 or so restrictionendonucleases. Thus it is exceptionallyfortunate when a linkage is found relativelyearly during familial studies, as recently seenwith IgE responsiveness and chromosome Ii.5In this instance a linkage was found when onlythe 17th gene probe and one restriction enzyme(Taq 1) were used.The process of genetic linkage depends on

the disease and an identified polymorphismcosegregating: individuals with disease showthe polymorphism whereas those without donot. But because genetic material may crossover during meiosis the polymorphism maybecome separated from the abnormal gene, andthis is more likely the further they are apart.Separation of the polymorphism from theabnormal gene and hence the disease is due torecombination. The proportion of siblings inwhom this occurs determines the recombinationfraction. For instance, if in 10 siblings five have

disease X and polymorphism Y, but only onesibling without the disease has polymorphismY, the recombination fraction is probably 1 outof 10 or 0-1. This value is used to describe howfar apart the gene for disease X and thepolymorphism Y occur in the genome. In thisexample the two loci are 10 centimorgans (cM)or map units apart.The next step consists of mathematical

calculations of the likely value of the re-combination fraction, which will depend onmany factors, including the number of siblingsstudied and the disease penetration. This issimple but tedious and is usually calculated bymeans of a computer program.6 The likelihood(or odds) that this recombination fraction iscorrect is conventionally expressed as itslogarithm-the logl0 ofthe odds or LOD score.The LOD scores for several families are then

added together and the sum is then plotted foreach recombination fraction. The maximumlikely estimate of the recombination fraction isthe value that corresponds to the peak of theLOD scores. Thus in the example above aLOD score greater than 3-0 means that theodds are more than 1000:1 that a linkage occursat this distance (10 centimorgans) between thegene and the polymorphic locus. In otherwords, the recombination fraction is very likelyto be close to 01 and hence the restrictionfragment length polymorphism to be within 10centimorgans of the abnormal gene.Once this association has been determined

with confidence it is then possible to getprogressively closer to the defective gene by theuse of overlapping probes.

Northern and dot blotting ofRNANorthern blotting is similar to Southernblotting, except that the single stranded DNAprobe is hybridised to complementary RNAmolecules that have been blotted on tonitrocellulose after separation by size.Alternatively, cellular RNA may be directly"dotted" on to nitrocellulose and hybridised tothe probe (dot blotting). These techniques maybe used to assess the tissue specificity of geneexpression. In addition, the Northern and dotblots can be semiquantified by densitometryscanning of the autoradiographs obtained, andthis has facilitated the study of factors that alterthe rates of gene transcription.The major problem in assessing changes in

the amount of RNA present in cells relates tothe possible variability in purification yield. Totake account of this possibility the results arecorrected for the total amount ofDNA orRNAthat has been transferred to the nitrocellulosemembrane. Alternatively, sample results areinterpreted by comparison with a gene thatshows constant expression (for example ,Bactin).

Oligonucleotide directed mutagenesisOligonucleotide directed mutagenesis is a tech-nique used to alter the gene being studied in aspecific way to gain insight into protein func-tion and even to produce designer proteins (see

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below). Any nucleotide in a fragment ofDNAcan be specifically mutated to one of the otherthree nucleotides. The method requires thattheDNA fragment to be mutated is available inpure single stranded form and is cloned into anappropriate vector. The next step is to syn-thesise chemically an oligonucleotide 7-20 basepairs in length that is complementary to thenatural DNA but has a single base mismatch.The oligonucleotide will hybridise to the nativeDNA under carefully controlled conditionsand can then be used as a template for primingDNA extension to form a heteroduplexmolecule. The heteroduplex is then trans-formed into a bacterium and the bacterialDNApolymerase subsequently "repairs" the basemismatch to yield either the original or themutant sequence. Further aliquots of theoligonucleotide sequence can be radiolabelledwith phosphorus-32 and then under morestringent conditions used as a probe to screenfor the mutant colonies. DNA sequencing isthen used to confirm that the procedure hasbeen successful.

Site directed mutagenesis has been used toproduce new proteins. Any alteration infunction will help to elucidate the primarystructure-function relationships of the protein.For example, site directed mutagenesis of thegene that codes for the beta adrenergic receptorhas shown that two cysteine residues onadjacent loops of the protein are important formaintenance of the tertiary structure (probablyby the formation of a disulphide bond betweenthe two loops) and hence the binding of ligandto the receptor.7 Furthermore systematic sub-stitution of neutral residues for acidic aminoacids within the ligand binding domain of thebeta receptor, by site directed mutagenesis ofthe gene, has resulted in identification of thespecific amino acid residues which bind to theprotonated amine group of the adrenergicligands.8

Site directed mutagenesis has also been usedto design new molecules for a defined purpose.For example, site directed mutagenesis of thereactive centre of al antitrypsin (Met 358 -+Val)has resulted in the production of a mutant a,antitrypsin species that is resistant to oxidativeinactivation but retains the elastase inhibitoryproperties of the native protein. This modifiedinhibitor has been considered to have thera-peutic potential in conditions where a reduc-tion in a, antitrypsin function due to oxidationis thought to be critical in the pathogenesis ofchronic diseases.9

Expression vectors and cell freetranslation systemsDNA from individuals with a naturally occur-ring mutation or DNA manipulated in vitro bysite directed mutagenesis may be introducedinto a cell to determine the effects of themutation on gene expression. The cells mostcommonly used are Xenopus (toad) oocytes andcells from the fertilised eggs or embryos ofXenopus laevis, the mouse, or the flyDrosophila melanogaster. Xenopus oocytes aremost often used because they are readily avail-able in large numbers. Each fully grown oocyte

is a large cell (0-8-1-2 mm) with a corres-pondingly large nucleus, thus making micro-injection ofDNA into the nucleus technicallyeasy. Single stranded DNA is converted into aduplex form in the oocyte nucleus and thenassembled into chromatin. The oocyte nucleuscontains eukaryotic RNA polymerases, whichtranscribe the exogenous DNA. When proteincoding genes are injected into the nucleus andcorrect initiation of transcription occurs stableRNA is produced, which is transported into thecytoplasm. TheRNA is translated into protein,which is then secreted by the oocyte into theincubation medium. In this way it is possible tostudy the effects of a mutation in a genesequence on transcription, translation, andsecretion of the protein product.The oocyte may also be used as a cellular

expression system for the study of mRNA.Injection ofmRNA into the cytoplasm leads toprotein synthesis, modification, and secretion.Alternatively, isolated mRNA species may bestudied in cell free translation systems (forexample, rabbit reticulocyte lysate or wheatgerm lysate systems). Again translation of themRNA occurs, leading to the formation of theprotein product.The mechanism for a, antitrypsin deficiency

associated with the homozygous ZZ state hasbeen partly elucidated by these techniques.Messenger RNA isolated from the liver ofnormal subjects and individuals homozygous(ZZ) for the a, antitrypsin defect has been usedin cell free translation systems and in theXenopus oocyte secretory system. The studiesshow that the low plasma concentrations of oa,antitrypsin are the result of a block in thetransport of the protein from the rough endo-plasmic reticulum through the secretory path-way and not the result of impaired synthesisfrom mRNA.'0

DNA sequencingThe development of techniques to determinethe sequence of DNA molecules has enabledrapid advances in our knowledge ofgene struc-ture and function to be made and has allowedthe defects underlying many genetic disordersto be determined. In addition, a knowledge ofthe basic sequence ofDNA is a prerequisite forsubsequent manipulations of DNA moleculesby site directed mutagenesis. Once a sequencehas been changed the sequence of the producthas to be checked to confirm that the requiredmutation has been obtained.The most widely used method for DNA

sequencing was developed by Sanger et all' anduses an enzymic dideoxynucleotide technique(fig 3). The DNA molecule to be sequenced isinitially cloned in a bacteriophage. A syntheticoligonucleotide with a sequence complemen-tary to about 15 bases of the phage sequence 3'to the inserted DNA acts as a primer for DNAsynthesis. The Klenow fragment of DNApolymerase is then used to synthesise a series ofDNA molecules complementary to the DNAbeing sequenced. The reaction is carriedout in the presence of dideoxynucleosidetriphosphates.

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5''CTACGT 3 3'Target Priming

sequence site

+ pnmer

15'PnPmer

+ radiolabelled nucleotides (d ATP, + d CTP, + d GTP, +

d TTP)+ Klenow fragment of DNA polymerase+ dideoxynucleoside triphosphate analogues (dd ATP,or dd CTP or dd GTP or dd TTP)

Synthesis of radiolabelled complementarycopies of the target sequence

dd GATGCA -Edd ATGCA Random incorporation of

ddTGCA ....> chain terminating analoguesresults in the generation ofdd GCA } radiolabelled fragments which

dd CA t vary in lengthdd A

Denaturation + gelelectrophoresis

Directionof

electrophoresis

A C G T -4 Dideoxy analogueused in incubation

-| mixture

AUTORADIOGRAPHOF GEL

sequence read3' GATGCA 5'

mixtures is then denatured and electro-phoresed in adjacent lanes. The radioactivebands ofDNA are detected by autoradiographyand the sequence can then be read directly fromthe autoradiograph as the DNA chain sequencewas stopped.

As stated at the beginning of the first article,these two introductory articles can do no morethan outline some of the principles, methods,and terminology used in molecular biology.With the increasing use of these techniques indiagnosis and pathogenesis it is becomingcritical for everyone with a scientific interest inrespiratory medicine to understand the basiclanguage ofthe science. We hope that these andthe subsequent articles in this series will makethe transition as painless as possible.

1 Southern EM. Detection of specific sequences among DNAfragments separated by gel electrophoresis. J Mol Biol1975;98:503-17.

2 Orkin SH, Little PFR, Kazazian HH, et al. Improveddetection ofthe sickle mutation by DNA analysis. NEnglJMed 1982;307:32-6.

3 Kidd VJ, Globus MS, Wallace RB, et al. Prenatal diagnosisof a, antitrypsin deficiency by direct analysis of themutation site in the gene. NEngl JMed 1984;310:639-42.

4 Kan YW, Dozy AM. Polymorphisms of DNA sequenceadjacent to human beta-globin structural gene: relation tosickle mutation. Proc Natl Acad Sci 1978;75:5631-5.

5 Cookson WOCM, Sharp PA, Faux JA, et al. Linkagebetween immunoglobulin E responses underlying asthmaand rhinitis and chromosome I lq. Lancet 1989;i:1292-4.

6 Lathrops GM, Lalouel JM. Easy calculations ofLOD scoresand genetic risks on small computers. Am J Hum Genet1984;36:460-5.

7 Dixon RAF, Sigal IS, Candelore MR, et al. Structuresrequired for ligand binding to the beta adrenergicreceptor. EMBO Journal 1987;6:3269-75.

8 Strader CD, Sigal IS, Register RB, et al. Identification ofresidues required for ligand binding to the beta adrenergicreceptor. Proc Nat! Acad Sci 1987;84:4384-8.

9 Courtney M, Jallat S, Tessier LH, etal. Synthesis in Ecoli ofa, antitrypsin variants of therapeutic potential foremphysema and thrombosis. Nature (Lond) 1985;313:149-51.

10 Verbanac KM, Heath EC. Biosynthesis, processing andsecretion of M and Z variant a, antitrypsin. J Biol Chem1986;261:9979-89.

11 Sanger F, Nicklen S, Coulsen AR. DNA sequencing withchain-terminating inhibitors. Proc Natl Acad Sci 1977;74:5463-7.

The Klenow fragment of DNA polymeraselacks the 5'-+3' exonuclease activity of theintact enzyme and thus can also use dideoxy-nucleoside triphosphate molecules as sub-strates. These analogues lack a 3' OH group andonce they have been incorporated into theDNA chain they can no longer act as a substratefor further chain elongation, so that the grow-ing DNA chain is terminated. Four reactionmixtures are set up, each with all fournucleotide triphosphates, one of which islabelled with phosphorus-32 and a low concen-

tration of one of the four dideoxynucleosidetriphosphates. This results in the production ofa population of partially synthesised radio-active DNA molecules each with a common 5'end and each varying in length to a base specific3' end as one of the dideoxynucleotidesbecomes incorporated. The use of four mix-tures means that a dideoxynucleotide should beincorporated into every nucleotide position inat least one of the lengthening sequences,thereby stopping the process. Each oftheDNAfragments produced will differ in size from theothers. The DNA in each of the reaction

Glossary

Anneal Joining together of two complementarystrands of nucleic acids by hydrogen bonding betweenopposite base pairs.Bacteriophage Bacterial virus that can be used as a

cloning vehicle.Centimorgan (cM) Unit of measurement for thedistance between two loci on a genetic map.Cloning Introduction of fragments of DNA intobacteria to produce millions of identical copies (clones).Cosmid Hybrid cloning vector composed of plasmidand bacteriophage, which is used to clone largefragments ofDNA.Denaturation Dissociation of two complementarystrands of nucleic acid by heating or treatment withalkali.Hybridisation See "Anneal."Klenow enzyme Fragment of the enzyme DNApolymerase that catalyses DNA synthesis but lacks the5'- 3' exonuclease activity of the original enzyme.Map unit (mu) See "Centimorgans."Northern blotting Technique for identification of thepresence and size ofmRNA.Oligonucleotide directed mutagenesis Technique inwhich a specific nucleotide of a gene is mutated forstudying the structure-function relationships ofcomplex proteins.Penetrance The frequency with which a gene mani-

Figure 3 Schematicrepresentation ofDNAsequencing (Sangermethod). The sequencethat is read off thesequencing gel iscomplementary to thetarget sequence.

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fests itself in the phenotype of an individual.Plasmid Self replicating circular, double strandedDNA molecule found in bacteria: the most commonlyused cloning vehicle.Polymorphism One of two or more alternativenucleotide sequences at a genetic locus.Probe Single stranded DNA or RNA used to identifycomplementary single stranded DNAs or RNAs incomplex mixtures of nucleic acids.Recombinant DNA molecule New DNA moleculeconstructed in vitro from two or more different DNAsequences.Restriction map Map of the location of all possiblecleavage sites for restriction endonucleases relative toeach other on the DNA being analysed.

Restriction endonuclease Enzyme that recognises a

specific short nucleotide sequence of DNA and cleavesthe DNA at or close to this site.Restriction fragment length polymorphism DNApolymorphism that results in either the loss or the gainof a restriction site for a restriction enzyme and hencealters the pattern of fragments obtained after digestionof the DNA with this restriction enzyme.Reverse transcriptase Enzyme that catalyses the syn-

thesis of single stranded complementary DNA (cDNA)with messenger RNA as the template.Southern blotting Technique to identify the presenceand size ofDNA molecules.Transformation Process in which extracellular DNA istaken up by a cell.

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Applications to study respiratory disease: methods biology