Sor gene isolation, However, a€ well as being wsefial Sor the tolation of speciic ragments, thePCReanhe ‘used to generate libraries. Le. by amplifying a repres: entative collection of random gensmnie fagments. ‘This can be achieves! using eller randoen primers Sllenwed by size selection Sor suitable PCR prowgucts orasirategy in which penumnic DNA js digested with restriction enzymes and then linkers are ligated to the ends ofthe DNA fragments, providing annealing, sites for one specific type of peimer fe-g, Hang ct al, 1992, Cheung & Nelwa 1996}, These techniques fare powerful Because they allow genic fragment Ubearies to be prepared from material tat cauld not ‘viel DNA of suitable quality er quantity for conven anal brary coastruction, burt competibioa among, the templates generally does nod allaw the produc. ton ofa truly representative lbeary. CDNA cloning Propertienaf cDNA DNA is peepared by reverse-transcribing exlfular RANA, Cloned eukaryotic eDNAs ave their awa special uses, which derive from the fact that they Jack introns and other non-coding sequences pre. seat in the corresponding genomic LNA. introns fare rare in bacteria but occur in most genes af higher eukaryotes, They can be situated within the ending sequetce itself where they then interrupt ‘thecollinearrelationshipofthe gene andits encexied polypeptide. cr they may accurin the Soe Vuntrans. lated regions kn any event. they are copied by RNA polymerase when the gene is transcribed. The primary transcript goes thromph a series of process ing events in the nuckus before appearing in the syloplasm as mature mRNA. These events include the removal of introm sequences by a process called splicing. In mammals, same genes contain numer: ‘our large intrans thal represent the vastmajrty af the sequence. For example, Uke human dystrophin genecontains 79 introns, representing aver 99% af the soquence. The gene is nearly 2.5 Mh in length sana yet the earresponding EDINA is only just ever 11 kb, Thus, one advantage of CDNA cloning is that in many cases thesiaeofthe eDNA clone significantly Jower than that ef the correspanding geomic cane. Since removal of cukaryotic intron transcripts by splicing does not occur in bacteria, eukaryotic cDNA ‘clones find application where bacterial expressiars of the foreign DNA is necessary, either asa prerequisite for detecting the clone (s0© p. 104) or because ox pression of the polypeptide product is the prirary ehjective. Alsa, where the sequence of the genale DNA isavailable, the pasition af introa/exon bound: aries can be assigned by comprise with the cDNA sequence, DNA libraries (Under samecincumtances, il may be possiblete pre- pare cDNA directly from a purified mRNA. spaces. Mach mare commonly, a cDNA library is prepared by reverse-transeribing a population afmBNAs and then screened for particular clones. An important ‘conceptis that the eDNA library is repeesentative of the RNA population from which it was derived. ‘Thus, whereas genomic libraries are essentially the same, regardless of the cell type or developmental stage from which the DNA was isolated, the con. tenis of cDNA libraries will vary widely according tothese parameters, A given cDNA library will alsa be enriched for abundant mitNAs but may contain only a fow clones representing rare miRNAs. Pure thermoce, where # gene is diflerentially spliced, a DNA library will contain diferent eleates represent ingalterativesplice variants. Table 6.2 shoms the abundances ofdllferent clases ef mRNAs in twa representative tissues. Generally. mRNAs can be described as abundant, moderately abundant er rare, Notice thal. in the chicken eviduct, one mRNA type is superabundart. This encodes ovalbumin, the major eggavhite pealein. ‘Therelore, the starting population & naturally sa enriched in ovalbumin mRNA that belating whe evalbumineDVA can beackicved without the wse of alibrary. An appropeiate strategy for obtaining such abundant cDNAs istoclone them directlyinan MI? vveclor, suchas M1 Simp. A sel of donescan thea be sequenced immediately ard dlentified om the busts efthe polypeptide that each encodes. A successful demonstration of this strategy was reported by Pulney eta. (1982), whodetennined DNA sequences 0117 randomly chosen muscle-clINA clones. Based en the aming acid sequences available fer 19 abundant muscle-specitic proteins, they were able taTable G.2 Absindanceclssesoftypical ARNA populazhs. Nester of “Abundaace ‘Source different miRNAs (melecules/eell) Mouse Boe cpoplaunic peta ad nea 11500 15 ‘Chek onctuerpeyaaeil pata 1 1o0000 7 4000 5 ‘Ratrence: usa (Yung ata 176 kde vie (ual ea. 157, identify clones corresponding ta 13 of these 19 pratems, including several protean variants. For the isolation of DNA clones in the moderate and low-abundance class, itis usually necessary to-construct a eDNA library. Once again, the high efliciency obtained by packaging in vilro wakes [Link] attractive Sor ebtainiig Large nur. bers of eDNA chines. Phage. insertion vectors are particularly well suited far CDNA cloning and sore of themast widely uscd vectorsarediscussedin fox 6.1. ‘Typically, 10°10" clones aresulficient far the tsa. Son [Link] mRNAs from most cell types, i.e, those present at 15 molecules per cell or abore. Howwever, ome mRNAs areeven lessabundamt than this, and may be further diluted ifthey are expressed inonly afew speciliceellsin aparticular tissue. Under these circumstances, it may he worth enriching the RNA preparation prior to library construction, ¢.¢- bby sie fractionation, andtesting the fractions for the Prewnce ofthe desired molecule. One way in whichthis can be achieved is toinject mRNA fractions into ‘Newopus oocytes (p. 215) and test them for produc: Hon of the corresponding protein (Melion 1987}. See abothe discussion of subtraction claringan p. Preparationof cDNA farlibrary construction ‘The cDNA syuthesis reaction “The synthesis af double-stranded cfINA suitable for insertion inte a cloning vector involves three major steps: (i) firsistrand DNA synthess on the mRNA template, curried out with a reverse transcriptase: (it) remaval of the BNA template; ar (ill} second strand DNA synthesis sing thefiret DNA strand aca template, curried out with a DNA-dependent DNA polymerase, such ax call DNA palymerase [All DNA polymerases, whether they use RNA or DNA as the template, require a primer to initiate strand synthesis, Development of (DNA cloning strategies “The first reports of cDNA cloning were published in the mid-197(tsand were all kased on the bemapeily- mer failing leclnique, which is described briefly in Chapter 3. Of several allemative methods, the one tut became the most popular was that of Maniatis al (19764, This involved the use of an aligodT primer annealing af the polyadenylate tail of the miRNA to prime first-strand cDNA syathesis, and tweik advamtage of the fact that the fist eDOA strand bs the tendency ta transiently fold bite om itself, forming a hairpin loop, cesulling in self priming of the second strand (Efstratindist al. 1976). After the synthesis of the second DNA strund, this loop must be cleaved with a single-strand-specific nuclease, ‘e.g SI nuclease, to allow insertion inte the cloning vectar (Fig. 6.5), A senour disadvantage of the Fsairpins method ks that cleavage with $1 nuclease results in the loss of a certain amount of sequence at the 5 end of the clone. This strategy bas therefore been superseded by other methods in which the second strand is primed ina separate reaction, One of te simplest strategies is shewn in Fig. 6.4 (Land et al. 1981). After fire-strand synthesis, which is primed with an coligoT primer as usual, the cDNA is tailed with a string of cytidine residues using the enzyme ter~ ‘nial transferase. This artilcial ipod tail is them ‘wed as an annealing site for a synthetic [Link]: ‘primer, alliwing synthesis of the second sirand. Using this method, Land et al. (1981) were able to ‘isolate a fulllength eDNA comesponding to the chicken lysazyme gene. However, the efficiency can ‘belower fur other cDNAs (e.g. Cooke rt al. 1980) For cDNA expression bbcaries, itis advantageous ifthe cDNA can be inserted into the vectar in the ‘correct orientation, With the self priming metho, ‘his can be achieved by adding a synthetic linker ta the double-stranded eDNA molecule before the ‘hairpin loop is caved (ep. Kurtx & Nicodemus 1981: Fig. 6.721, Where the second strand i primed separately, direction cloning can be achieved using an oligodT pemer containing a linker sequence (e.g. Coleclouph & Erlit 1985; Fig.6.7b). Analtern. alive isto use primers for eDNA synthesis that are already linked ta plasmid (Fig. 6.7. This strategy ‘was devised by Okayama and Blerg (1982) and has ‘wo further notable characteristics, Fest fullengthe cDNAs are preferentitly shusined because an RNA- DNA hybrid molecule, the result of firststrand synthesis isthe substrate fer a terminal transferase reaction. cDNA that dues not extend to tte end of ‘themRNA will present ashiekled Sohydroxyl group, ‘which is a poor substrate far tailing. Secondly. the ssecomd-sirand synthesis step primed by nicking the BNA at multiple sites with RNase H. Second-sirand synthesis therefore occurs ky a nicke-translation ‘ype of reaction, which ts highly efficient. Simpler cDNA cloning strategies incarparating replacement synthesis of the secoad strand are widely used (e.g. Gubler & Hallinan 1983, Lapeyre & Amalric 1985). ‘The Gubler-Hoifiman reaction, ax it ts commanly “known, is show in Fig. 6.8. Full-length cDNA cloning Limitations of conventional cfoning strategies Conventional approachesta the praductionefeDVA Ubsaries have inva major drawbacks. First, where ‘oligo-dT primers are used to initiate first-strand syu- ‘thesis, there is generally 3'-end bins (preferential recovery of clones cepresenting the ¥ end of cDNApc sere: Perce o: Sate framiermation hast repair. wee] § Soe (eA — Fig. 6.5 sts eary DNA ding ra lservahe eta aaethe yee Javon ipl seu eran DNA sys adapts lt