Capture PCR: Efficient Amplification of DNA Fragments Adjacent to a Known Sequence in Human and YAC

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Capture PCR: Efficient Amplification of DNA Fragments Adjacent to a Known Sequence in Human and YAC DNA Maria Lagerstr6m, J~iri Parik, Helena Malmgren, Jason Stewart, UIf Pettersson, and UIf Landegren

Department of Medical Genetics, Uppsala Biomedical Center, Uppsala University, S-751 23 Uppsala, Sweden

We have devised a procedure, and provide access to DNA se- ing DNA segment. The oligomers that termed capture PCR (CPCR), that quence information for these end are added to all termini may be permits the rapid isolation of DNA fragments in a minimal number of designed so that a complement for the segments situated adjacent to a steps. With the aid of the present general primer is formed only after a characterized nucleotide sequence. technique, we have isolated over molecule has been copied from a In this procedure, a DNA sample is 100 end fragments from YACs specific primer hybridizing internal to restriction-digested and a linker, derived from the human X chromo- the fragment. This is done to avoid comprising two base-paired oligo- some. Isolated end sequences have amplification of all DNA sequences nucleottdes, is added to the ends by been used to order YAC clones into surrounded by oligomers. For instance, ligation. Multiple extension reac- a contig. in the vectorette strategy, or bubble tions are performed using a biotiny- PCR, (8,9) a duplex oligonucleotide with lated primer derived from the an internal mismatched segment must known sequence, permitting the be copied before a complement for the subsequent isolation of extension general primer is formed. Still, in this products on a streptavidin-coated technique, unlike conventional PCR, support. The enriched fragments The PCR technique provides for the priming by a single oligonucleotide is are amplified exponentially using highly specific amplification of unique sufficient to generate a template that another specific oligonucleotide, DNA segments defined by two sur- can subsequentially undergo exponen- hybridizing 3' to the biotinylated rounding primer sequences. ~1~ A num- tial amplification, greatly increasing primer in combination with one of ber of procedures have been developed the potential for false reaction prod- the linker oligonucleotides, now to apply PCR in circumstances where ucts. functioning as a PCR primer. The sequence information is available from In contrast, techniques relying on convenience of CPCR is greatly en- only one side of a segment of interest. the hybridization of two specific hanced by using a novel strepta- For example, in inverse PCR, DNA frag- primers afford much greater specifi- vidin-coated manifold, which is con- ments are ligated into circles. ~2-4~ Thus, city. C1°-14t Mueller and Wold described structed so that it projects into a known DNA sequence can be made the use of a specific primer for a first each individual well of a microtiter to surround sequences normally lo- extension reaction in fragmented DNA plate. The procedure permits the si- cated on both sides of this DNA seg- to generate a blunt end that is then multaneous isolation of fragments ment, permitting their amplification ligated to a blunt oligonucleotide from large numbers of DNA using defined primer sequences. A linker. (1°~ In a subsequent reaction, samples and minimizes the risk of series of other methods, sometimes this fragment is amplified using a contamination between reactions. termed anchor PCR, ~5-9i avoid the re- linker-primer and a specific primer, lo- We have applied this method to quirement for circularization by the cated downstream of the one used in identify DNA sequences located addition of oligomers to all ends in a the first extension reaction. This tech- downstream of known sequences in fragmented DNA preparation. These nique has proven sufficiently specific the human genome. The technique oligomers may be added by enzymatic to permit the sequencing of single- has also been used to identify end tailing, ~5,61 or by ligation,/7~ and can copy gene sequences directly from fragments of sequences cloned in a then be used as complements for a genomic samples. ~11 yeast artificial chromosome (YAC) general amplification primer. This The number of different techniques vector. The reactions can be in- primer is used in conjunction with a for amplifying DNA segments bordered itiated directly from yeast colonies specific primer to amplify an interven- on one side only by known sequences

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attests to the importance of the prob- port permits the simultaneous process- Capture PCR lem as well as to the lack of a clearly ing of large numbers of reactions and Genomic DNA was digested with superior technique. Existing methods greatly reduces the potential for con- restriction enzymes and, in the same exhibit poor specificity or are cumber- tamination between samples. Further- reaction, linkers were ligated to the some to perform. We now present a more, we show that, with a standard DNA fragments (Fig. 1). To avoid con- simple and highly specific method to set of oligonucleotides, end fragments tamination, tips with barriers (Aerosol isolate sequences located adjacent to a from YAC vector inserts may be isola- Resistant Tips, Continental Laboratory characterized DNA segment based on ted and sequenced using a rapid Products) were used for pipetting the hybridization of two specific protocol starting directly from yeast throughout the protocol. All the reac- primers. In this method, a biotiny- colonies. tions were performed in flexible micro- lated, specific primer is used for a first titer plates (Falcon 3911). For the com- set of extension reactions in DNA bined restriction and ligation reaction, modified by the addition of oligonu- 100 ng of DNA was incubated with a cleotide linkers to all restriction ends. METHODS pair of oligonucleotide linkers (1°) Extension products, enriched for the Reagents (Table 1) at 2 ~tM each, one end of sequences of interest, are captured on a Oligonucleotides were synthesized by which can join to restriction ends novel streptavidin-coated support. The standard phosphoramidite chemistry. without recreating the recognition se- relevant fragments are then amplified The sequences of the oligonucleotides quence. One-half microliter of restric- with a primer representing the oligo- employed are given in Table 1. A 5' tion enzyme (usually approximately 5 nucleotide linker and a primer hybrid- biotin residue was incorporated by units, depending on the concentra- izing downstream of the biotinylated reacting a hydroxysuccinimide ester of tion) and 0.5 ~tl of T4 DNA ligase (4 oligonucleotide. This principle of biotin (Clonetech) with an oligonucle- units, Pharmacia) were added to a final physically enriching extension prod- otide having a primary amino group volume of 10 ~tl containing 0.5 mM ucts using a biotin-streptavidin inter- added as an amino-linker residue. The ATP, 10 mmoles Tris-HC1 (pH 7.5), 10 action for subsequent amplification reaction products were purified by mmoles MgCl z, and 50 mmoles potas- has been developed independently by FPLC over a reverse-phase C18 column. sium acetate. The reaction was in- Rosenthal and Jones.O4) We demon- Alternatively, a biotinylated phosphor- cubated at 37°C for 3 hr. strate in this paper that the technique amidite residue was directly added dur- When intact yeast cells were used is of sufficient specificity to identify ing oligonucleotide synthesis (Biotin as a source of DNA, a single colony was unique sequences in the human dX, Midland Certified Reagents Co, resuspended in 50 ~tl of H20. Five mi- genome. The streptavidin-coated sup- TX). croliters of the suspension was added

TABLE I Sequence of Oligonucleotides Used in the Present Work

Target sequence Oligonucleodde Nucleotide sequence

Oligonucleofidesligatedto msmcfionends: linker 1 5' GCGGT GACCC GGGAG ATCTG AATTC 3' B-linker 1 B 5' GCGGT GACCC GGGAG ATCTG AATTC 3' linker 2 3' C TAGAC TTAAG 5' linker 3 3' C TAGAC TTAAG GC 5' linker 4 3' C TAGAC TTAAG CTAG 5' Specific capture and amplification oligonucleotides:

Amelogenin BAM9 B 5 GCCCT CCAGA TATTG CTGGT CTTA 3' AM11 5 TTTTT GTGGT TGCCC GGGAT CCTGC 3'

ZFX B242 B 5 CGTCA CCCGT CAGAG CTCAA 3' 244 5 AAAGC ACATG AGAAT CCATA 3' 241 5 TAGAA GAGTC TGCAG ACCTA TATTC 3'

~globin BI31 B 5 ATGGT GCACC TGACT CCTGA 3' 161 5' TGA GGAGA AGTCT GCCGT TA 3' 166 5' AGA AGTCT GCCGT TACTG CC 3'

YAC left BYAC8 B 5 TCGGA GCACT GTCCG ACCGC TTTG 3' YACI8 5 GAACC ATCTT GGAAG GACCG GATAA 3' YACII 5' GAATC CGCGG TAGCC AAGTT GGTTT AA 3' YACI6 5 TGTAA AACGC GGCCA GTCGG TAGCC AAGTT GGTTT AAGG 3' YAC6 5 GACTG TAATT TATCA CTACG 3'

YAC right BYAC9 B 5 ATGCC GGCCA CGATG CGTCC GGCG 3' YAC2 5 CAAGT CTGGG AAGTG AATGG AGAC 3' YACI 5' C CCGGG GGCGA GTCGA ACGCC CGAT 3 ° YACI7 5' TGTAA AACGA CGGCC AGTTC CCGGG GGCGA GTCGA ACGCC CGA 3' YAC5 5' CGAGT CGAAC GCCCG ATCTC AAGAT TA 3'

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to a 20-~tl standard PCR reaction con- primer (Table 1). One-half microliter of taining 0.1 ,uM biotinylated primer. product from the original PCR was 7 Restrict and ligate The temperature was varied between used as template in a 50-~tl PCR with 1 II 94°C for 1 rain, 55°C for 1 rain, and ~M each of the internal primer and Ii 72°C for 2.5 min in 35 cycles followed linker 1 (Table 1). The reaction produc- ~:7 Extend by a final incubation at 72°C for 7 ts were analyzed by agarose gel elec- IIIi:!:!:.~ rain. trophoresis to identify fragments exhi- The biotin-labeled extension prod- biting the expected change in size ducts were immobilized on a commer- upon using the internal primer. (2) ~7 Capture cially available polystyrene device, Samples from the original PCR were configured as a microtiter plate lid separated in an 1.5%o agarose gel and with eight rows of 12 pin-and-ball ex- appropriate fragments excised from the 7;7 Amplify tensions projecting into individual mi- gel and diluted in 200 B1 of H20. After li:i:i:~ crotiter wells (Falcon 3931). A number melting at 95°C, 1 ~tl of the samples I m of means of covalently coating the 96 was used as templates in a 50-~tl PCR, FIGURE 1 A schematic description of cap- prongs of the support with streptavidin using the specific primer together with ture PCR and of the solid support used. were examined and will be presented a primer representing sequences lo- Linker oligonucleotides are shown in black, elsewhere 0. Parik et al. in prep.). A cated downstream of the specific and primers specific for a given genomic preliminary protocol, resulting in a region are dotted. Genomic I)NA is digested primer in the opposite orientation. (3) with a restriction enzyme, and, in the same binding capacity of around 1 pmole of The CPCR fragments were digested reaction, a linker is ligated to the cleavage biotinylated product per prong, is with restriction enzymes. Twenty-five site. The DNA sample is subjected to exten- available upon request. Before use, the microliters of the reaction product sion reactions under PCR conditions, using support was briefly incubated in a from the original PCR was incubated a 5' biotinylated primer. The reaction pro- washing solution of 0.1 M Tris-HCl (pH with 40 units of a restriction enzyme, ducts extend from the primer across the 7.5), 1 M NaCl, and 0.1% Triton X-IO0, known to cut downstream of the I)NA fragment of interest and terminate in and then incubated for 3 hr at room specific primer, in a 3-hr incubation at a sequence complementary to the added temperature in the wells of the exten- 37°C. The digested samples were sepa- linker. Biotin-labeled extension products are sion reactions. As an alternative, 40 ~tl rated in a 1.5% agarose gel. captured on a 96-pronged streptavidin- of prewashed streptavidin-coated mag- coated mainfold projecting into individual microtiter wells. After washes, a PCR is per- netic particles (Dynal A.S., Norway) Southern Blot Analysis of YACs formed using as templates the isolated ex- were added to the extension reaction DNA segments isolated by CPCR from tension products bound to the support. A and incubated at room temperature. ends of inserts in YACs were labeled by second specific primer, hybridizing down- The solid supports were subsequently random priming (Oligolabelling kit, stream of the biotinylated oligonucleotide, washed in individual microtiter wells Pharmacia). The labeled ends were is used as a PCR primer together with the once with washing solution, once with prehybridized with fragments from the linker oligonucleotide. The PCR yields a a denaturing solution of 0.01 M NaOH, YAC vector, amplified using the product containing the DNA segment of in- 1 M NaC1, 0.1% Triton X-IO0, and oligonucleotides YAC2 and YAC18 terest. again with washing solution. Finally, (Table 1), to ensure that vector-derived the solid supports were washed in PCR portions of the probes did not contrib- buffer. ute to the hybridization signal. The to a final volume of 10 ~tl containing The immobilized extension produc- probes were hybridized to blots 0.1 ~tM biotinylated YAC vector primer ts were used as templates in a PCR prepared from genomic DNA from a in a standard PCR reaction mix of 50 reaction. The streptavidin-coated set of YAC clones and the yeast wild- mM Tris-HC1 (pH 8.3 at room tempera- device was present during the entire type strain AB1380, digested with ture), 50 mM KCI, 1.5 mM MgC12, 200 PCR. If streptavidin-coated paramag- HindIII, and separated by agarose gel BM each of dATP, dCTP, dGTP, and netic beads were used, 2.5 ~tl of immo- electrophoresis.(tsl dTTP, 12.5 ~tg bovine serum al- bilized extension products, resus- bumin/ml, and 0.035 units/B1 of pended in 10 ~tl of PCR buffer, were Sequence Analysis of Fragments AmpliTaq (Cetus). The sample was employed as template. The 50-~tl PCR Generated by CPCR overlayered with mineral oil and dena- reactions contained the oligonucleo- The nucleotide sequence of CPCR pro- tured at 93°C for 5 min, followed by tide linker 1 and a specific primer ducts was obtained by solid-phase se- annealing at 55°C for 1 rain, and ex- (Table 1), representing a sequence |o- quencing (161 and analyzed by auto- tension at 72°C for 3 min in two cycles cated downstream of the biotinylated mated fluorescent sequencing, One- before proceeding with the restric- primer, at 1 ~tM each. The temperature half microliter of the product of the tion-ligation step as described above, was varied as described above. original PCR was amplified in a 50 ~tl but in a final volume of 20 ~tl. The identity of amplified DNA se- PCR with 0.1 ~tM linker 1, modified by Multiple rounds of extension reac- quences was confirmed by three dif- the addition of a 5 ' biotin group, and tions were performed from a biotiny- ferent methods: (1) A new PCR was 1 ~M of a specific primer with the M13 lated primer. One-half microliter of the performed using a specific primer lo- forward primer sequence incorporated restriction-ligation reaction was added cated downstream of the previous PCR in the 5' end (see Table 1). The PCR

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was performed for 30 cycles. Twenty different short complementary oligo- The primers were derived from the hu- microliters of the reaction was in- nucleotides to modify ends generated man amelogenin, ~17/ ZFX, t18~ and f~- cubated with 20 ~tl of prewashed by more than 30 different restriction globin genes. ~191 The amelogenin reac- streptavidin-coated paramagnetic enzymes. The short oligonucleotides tions are shown directly after CPCR, beads for 30 rain at room temperature. have low GC content, ensuring that whereas ZFX and j~-globin fragments The bound PCR products were they will not function as primers in were first subjected to gel purification resuspended in 100 ~l of 0.1 N NaOH, PCR reactions. The oligonucleotide and then analyzed as detailed below. 1 M NaCl for 15 min at room tempera- dimers are designed so that the liga- The identity of the isolated amelo- ture, followed by three washes in the tion of dimers to restriction ends will genin fragments was confirmed by same solution and one in l O mM Tris- not regenerate the recognition se- reference to a restriction map. 117/CPCR HCI (pH 7.5). The final wash was per- quence of the restriction enzymes. products of the correct size were ob- formed with 100 ~l 80 mM Tris-HCl Therefore, restriction digestion and tained for each of the four investigated (pH 8.9), 20 mM ammonium sulfate, ligation can be performed simulta- restriction enzymes that cut within 2 and 5 mM MgCI 2 and the samples were neously. This ensures the gradual ac- kb downstream of the CPCR primers resuspended in 6 ~tl of this buffer. The cumulation of the desired restriction (Fig. 2 and data not shown). The [~- sequencing reactions were performed fragments terminated by a standard globin- and ZFX-related fragments in 30 temperature cycles with fluores- 25-met sequence. The linker-modified were excised from an agarose gel, cent labeled M13 forward primers, DNA samples can be saved and stored diluted, and subjected to another PCR using the Taq Dye Primer Cycle Se- for later use with any set of two reaction. For fragments amplified using quencing Core Kit (Applied Bio- specific CPCR primers. primers derived from the ZFX gene, the systems) according to the manufac- Aliquots of the linker-modified downstream CPCR primer was applied turer's protocol. The four PCR reac- DNA samples were mixed with a together with a primer located inter- tions with differently dye-labeled M13 specific, biotinylated primer and sub- nally to the fragments and in the op- primers and each of the four different jected to 35 cycles of extension reac- posite orientation. All four identified dideoxynucleotides were pooled and tions, interfoliated with denaturation fragments were shown to contain in- precipitated. The dried pellet was and annealing steps. Biotinylated ex- ternal sequences hybridizing to this resuspended in deionized formamide tension products were captured on a new primer, establishing that the frag- and 50 mM EDTA, 5:1 (vol/vol), and 96-pronged, disposable support pro- ments are all derived from the ZFX loaded on an automated DNA Sequen- jecting into individual microtiter wells. gene or from a closely homolo-gous se- cer (Applied Biosystems, Model 373A). Streptavidin, covalently coupled to the quence. For [3-globin-related fragments, solid support, efficiently immobilized a primer located somewhat down- RESULTS the biotinylated molecules. The device stream of the second CPCR primer was Isolation of Human DNA Sequences permits the simultaneous washing and applied in conjunction with the linker by CPCR transfer of sets of 96 reaction interme- oligonucleotide. As can be seen in Fig- ure 2, the fragment obtained from the PCR achieves its high specificity as a diates between microtiter plates. Fur- [3-globin gene after AluI digestion and consequence of the requirement that thermore, it greatly reduces the risk of two oligonucleotide primers must contamination between reactions. one of the DraI fragments underwent hybridize under relatively stringent Commercially available streptavidin- the expected slight change of size upon amplification with a PCR primer lo- conditions in the appropriate orienta- coated paramagnetic particles may also tion near each other for an exponen- be used to capture biotinylated exten- cated internally. The other DraI frag- tial amplification to result. ¢1J We ex- sion products, but the handling of ment failed to give rise to the expected amined the possibility of obtaining the large numbers of samples is less con- amplification product and was scored same specificity by physically isolating venient and increased care has to be as an incorrect amplification product. extension products from one specific taken to avoid contamination between An AluI site was observed at the ap- primer and subsequently using these reactions. propriate location in the restriction enriched fragments as templates for After washes, extension products map for the [3-globin gene region. In PCR by applying another, downstream bound to the supports were transferred contrast, no DraI site was seen. How- primer in conjunction with a general to another set of microtiter wells. They ever, the [3-globin primer sequences are primer. were used as templates in a PCR reac- quite well conserved in the ~-globin First, stocks of modified genomic tion containing a primer hybridizing gene and a DraI site is present at the DNA were prepared by ligating linkers, downstream of the biotinylated one appropriate location, downstream of made up of two base-paired oligonucle- and another primer, representing the the 8-globin gene. Therefore, it is likely otides, to ends generated by restriction linker sequence added to restriction that the observed DraI fragment is digestion (Table 1). The oligonucleo- ends in the genomic DNA sample (Fig. derived from the ~-globin gene. tide linkers lack 5' phosphate groups, 1). The amplification products were in- Thus, CPCR is equivalent to con- ensuring that only one of the oligonu- spected after agarose gel elec- ventional PCR in specificity and it may cleotides is covalently joined to the trophoresis. Figure 2 demonstrates an be performed using simple steps and ends of the genomic DNA fragments. array of fragments, defined on one side under standardized conditions. As il- The 25-mer oligonucleotide linker 1~1°i by a primer sequence and on the other lustrated here, the technique may be can be used together with one of three by the presence of a restriction site. applied to restriction map a segment of

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quently, more than a single amplification product is seen upon agarose gel electrophoresis. This could be a consequence of partial digestion during the restriction-ligation step of CPCR or it could result from illegitimate priming events generating shorter end fragments or completely irrelevant amplification products. Authentic fragments are easily identified using an internally located, vector-derived primer. Figure 4 demonstrates the identification of CPCR products as true end fragments by observing the expected difference in FIGURE 2 Human DNA fragments amplified by CPCR using pairs of primers located in size between agarose-separated CPCR tandem. (Lanes I and 2) Amelogenin gene sequences amplified after digestion of genomic products, amplified with amplification DNA with Alul and Dral, respectively. (Lanes 3-10) Fragments obtained using primers derived primers located at different distances from the ZFX gene after digestion with Dral, Hincll, Nael, and PvulI (lanes 3, 5, 7, and 9, respectively). The identity of the observed fragments was investigated in the adjacent lanes 4, from the cloning site. Moreover, dif- 6, 8, and 10 by determining if the excised bands could serve as templates, generating an ap- ferent restriction enzymes generate end proximately 100-bp amplification product, in a PCR with primers located internal to authen- fragments of different lengths. Shorter tic products. (Lanes 11-16) CPCR products generated using primers derived from the (~-globin fragments define restriction sites that gene. A gel-purified product generated after digestion with Alul was reamplified using the should be located internally to the same primers (lane 11) or using a specific primer located 5 nucleotides downstream of the longer fragments if these are derived previous one (lane 12). Similarily, two products observed after DraI digestion followed by from the same sequence. As illustrated CPCR were reamplified using the same primers (lanes 13 and 15) or with the downstream in Figure 4, the presence of such one (lanes I4 and 16, respectively). The lanes are flanked by a 123-bp ladder (BRL) serving as size markers (lane m). restriction sites may be confirmed by restriction analysis of longer fragments. genomic DNA and to isolate fragments fragments from YAC inserts provides Rapid Preparation of Yeast DNA of interest. for rapid genomic walks by strides of Samples for CPCR several hundred kilobases. ~2°) In CPCR large numbers of samples may YAC End Fragment Isolation by End sequences may be obtained be handled in parallel and processed in CPCR from the inserts of all YAC clones rapid steps. The purification of DNA CPCR is well suited for applications using the same standard set of CPCR from individual YAC clones thus be- where large numbers of DNA samples primers (Table 1). To ensure that the are searched for DNA segments located procedure does not isolate sequences adjacent to a standard sequence. One present in the normal yeast genome, example is the identification of frag- the biotinylated primers, hybridizing ments located immediately internal to on either side of the YAC vector clon- vector sequences in YACs. YAC vectors ing site, were derived from pBR322 se- represent valuable cloning vehicles quences in the vector. Figure 3 demon- that can accept more than a megabase strates agarose gel-resolved end frag- of genomic DNA. Identification of end ments from a set of YAC clones. Fre-

FIGURE 4 Confirmation of the identity of amplification products obtained from YAC clone XY316 using CPCR primers derived from the right-hand side of the vector. The nonbiotinylated vector-specific primer used for the samples separated in lanes 1, 3, and 5 hybridizes 90 bp upstream of that used in lanes 2, 4, and 6. Lanes 1 and 2 reveal CPCR products obtained after digestion with AluI. FIGURE 3 Products of CPCR exemplifying the routine isolation YAC end fragments from a Lanes 3 and 4 represent Rsal digestions of series of YAC clones. (Lane 1) CPCR from a left YAC end using the restriction enzyme BclI; the samples in lanes 1 and 2, respectively. (lanes 2-14) CPCRs from right YAC ends and using the enzymes BclI, BglII, Bglll, HinPl, The CPCR reaction products in lanes 5 and HinPI, BstUI, DraI, DraI, EcoRV, EcoRV, HinclI, PvuII, and PvulI, respectively. 6 were generated after RsaI digestion.

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comes a rate-limiting step. Therefore, basis of sharing a marker DXS304. This fragment ends with the dinucleotide we investigated the possibility of per- marker is located in the vicinity of the AG, representing a half-site of the forming the isolation of YAC end frag- fragile X locus at the distal end of the restriction enzyme AluI used to genera- ments directly from individual yeast long arm of the X chromosome. ¢21/ te the fragment, immediately followed colonies after minimal preparation. End fragments, isolated by CPCR, were by the complement of the linker 1 se- Yeast cells were directly added to mi- used to probe DNA blots prepared from quence. The method used is generally crotiter wells, overlayered with mineral the relevant yeast clones and wild-type applicable for the sequence analysis of oil, and heated to 94°C for 5 min fol- yeast as shown in Figure 6A. As ex- fragments isolated by CPCR. lowed by primed synthesis of a new pected, overlapping YAC inserts have strand from a vector-derived primer at least one end hybridizing to the DISCUSSION using Taq polymerase and nucleotides. other in a reciprocal manner. The size In this paper we describe an efficient of the fragments detected on blots dif- The primer used was identical to that technique for the PCR-assisted isola- fers between the clone from which the later required in the extension reaction of DNA segments located adjacent fragment was isolated and overlapping tions from a biotinylated primer. The to a known nucleotide sequence. The clones because the end fragments are incubation at 94°C serves to make the procedure employs two specific interrupted by vector sequences. The yeast DNA accessible for the sub- primers, oriented in the same direc- hybridization pattern allows deduction sequent enzymatic reactions while in- tion, applied sequentially but under of the order and orientation of the hibiting nucleases and proteases. The standard PCR conditions, i.e., brief an- YACs with respect to one another as extension reaction renders the DNA nealing and extension at high indicated in Figure 6B. Moreover, the segment 3' to the primer double temperature. Extension products from analysis establishes the order of the stranded, as is required for the sub- the first primer are captured on a sup- isolated end fragments along the chro- sequent restriction digestion and liga- port before proceeding to an exponen- mosome, providing a physical map of tion. Figure 5 illustrates that direct tial amplification reaction using a end-isolation from yeast colonies gen- this region. This orientation of the downstream specific primer in con- erates results similar to those obtained YAC clones has been established inde- junction with a general primer. The with purified DNA, significantly reduc- pendently (M. d'Urso and D. Schles- specificity of the technique is similar ing the turnaround time in a YAC singer, pers. comm.). to conventional PCR permitting isola- walking procedure. tion of human single-copy sequences. Sequence Analysis of YAC End Fragments of up to 2 kb have been iso- Hybridization Analysis of the Fragments lated and only small amounts of Relative Orientation of a Set of YAC purified I)NA are required in the proce- Clones Isolated YAC end fragments represent a convenient source of sequence tagged dure. Indeed, denaturation followed by Isolated YAC end fragments may be sites, (22~ appropriately spaced in the an extension reaction is sufficient to used to identify YAC clones with par- genome. Furthermore, the availability render the relevant DNA segment ac- tially overlapping inserts by applying of nucleotide sequence information for cessible for analysis in a crude sample the fragments as hybridization probes. the end fragments permits PCR-assisted of cells. We used end fragments from three screening of YAC libraries to identify Central to the present strategy is YAC clones to orient these with respect overlapping YAC clones. ~23) We have the concept of enriching for molecules to one another. The three clones were applied a solid-phase fluorescent se- extended from a biotinylated primer. isolated from a YAC library on the quencing protocol to permit the rapid Biotinylated extension _products may acquisition of sequence information be captured using commercially avail- for newly isolated end fragments. able streptavidin-coated paramagnetic CPCR products of interest were diluted particles. (14) We have, however, experi- 3 4 and subjected to one more amplifica- enced great difficulties handling more tion reaction using a 5' biotinylated than just a few DNA samples at a time version of linker 1 and a YAC vector- using paramagnetic particles as a solid specific primer including the M13 for- support. Contamination between reac- ward sequencing primer as a 5' addi- tions becomes increasingly difficult to tion (see Table 1). The amplified prod- avoid when more than just a few reac- ucts were immobilized on strepta- tions are performed in parallel. In con- vidin-coated magnetic particles, dena- trast, the solid support used here tured and washed, and subjected to permits rapid washes and transfer of multiple rounds of Taq polymerase- sets of up to 96 reaction intermediates FIGURE 5 Comparison between YAC end assisted extension reactions in the between different enzymatic milieus isolation by CPCR starting from yeast presence of fluorescent-labeled M13 with little potential for contamination. colonies (lanes I and 3) or from purified This is of particular value in applica- DNA (lanes 2 and 4). Lanes I and 2 forward sequencing primers. Reaction represent the left end fragment from YAC products were loaded onto an auto- tions involving PCR, where purity clone XY492 and lanes 3 and 4 represent mated DNA sequencer. Figure 7 il- rather than large binding capacity is of the right end fragment from YAC clone lustrates sequence data from the end of the greatest importance. It is likely that XY669. a 272-bp CPCR product. The YAC end a capture step will be of general value

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A) ¥ACA YAC B left ,~!:gbt,. , : !eft M,gh}

L~ '= ' []R . • ~

FIGURE 6 Ordering of YAC clones by hybridization of isolated end fragments to Southern blots prepared from HindlII-digested DNA from the same yeast clones and from AB1380, a yeast strain not harboring any YAC. (A) The results of the hybridization analysis. (B) The relative map order of the YAC end fragments inferred from the hybridization analysis. in situations where currently nested proven valuable in several other prepa- samples. Since the same set of primers PCR is used to obtain increased rative and analytical molecular genetic is used for all yeast clones, the reaction specificity. The capture of specific techniques and it is particularily help- conditions may be completely stan- target sequences by hybridization to a ful in diagnostic applications (M. dardized. We have used CPCR to isola- biotinylated primer may also serve as a Samiotakis, unpubl.). te a total of more than 100 YAC end means to enrich rapidly for relevant se- The use of CPCR to isolate end frag- fragments. Using the two blunt-end- quences to permit specific enzymatic ments from DNA segments cloned into generating enzymes, AluI and RsaI, reactions starting from a crude tissue YAC vectors illustrates an important YAC end fragments were successfully sample. The possibility of handling application of the technique. Here, a isolated from both ends of approxi- large numbers of samples using the standard set of oligonucleotides may mately 85% of the YAC clones investi- streptavidin-coated solid phase has be used to process any number of DNA gated. With an extended set of restric-

A G T C T T G C T A G C G G T C T A T C A A T T T T G T T G A T C C T T T C A A A A A A C T A G O A A T T C A O A

!' 1

, I FIGURE 7 Results from a solid-phase, fluorescent DNA sequence analysis of a YAC end fragment isolated by CPCR from a YAC clone, digested with Alul. The illustration represents 57 bases of the nucleotide sequence toward the end of the CPCR product. The arrow denotes the begin- ning of the complement of the sequence of linker 1.

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tion enzymes, no YAC clone has intron-exon boundaries, or break- reaction to amplify cellular DNA proven recalcitrant to this procedure, points of deletions and translocations adjacent to an integrated provirus. J. although the presence of repeated se- may be identified using CPCR. By a virol. 63: 1924-1928. quences may limit the usefulness of a simple modification of the standard 5. Loh, E.Y., J.F. Elliot, S. Cwerla, L.L. given end fragment in genomic map- protocol, CPCR reactions could be per- Lanier, and M.M. Davis. 1989. ping. formed using randomly fragmented Polymerase chain reaction with single The speed with which a YAC walk- DNA samples, modified with the ap- sided specificity: Analysis of the T cell ing project may be conducted using propriate linker sequence. Amplifica- receptor delta chain. Science 243: CPCR is enhanced by the fact that tion products in a desired size range 217-220. multiple YAC clones may be studied in could then be selected after size separa- 6. Frohman, M.A., M.K. Dush, and G.R. parallel and the reaction may be in- tion. The CPCR strategy should also be Martin. 1988. Rapid production of ititated directly from a YAC clone useful for the isolation of populations full length cDNAs from rare seeded on a petri dish without DNA of DNA segments located adjacent to a transcripts using single gene specific purification. sequence present in multiple sequence oligonucleotide primer. Proc. Natl. Alternatively, the reaction may also contexts in a DNA sample. An example Acad. Sci. 85: 8998-9002. be initiated from cells scraped from a of this would be the definition of mul- 7. Roux, K.H. and P. Dhanarajan. 1990. frozen glycerol stock of yeast cells (data tiple viral integration sites in a sample A strategy for single site PCR not shown). Therefore, it is possible to of genomic DNA. Thus, the CPCR pro- amplification of dsDNA: Priming obtain end fragments from a large cedure represents a generally useful digested cloned or genomic DNA group of identified YAC clones in 1 or way of applying PCR in contexts where from an anchor-modified restriction 2 working days. The actual hands-on sequences outside of a known DNA site and a short internal sequence. time per set of 96 reactions is only a segment are sought. Biotechniques 8: 48-57. few hours. Moreover, isolated end frag- 8. Riley, J.H., R. Butler, D.J. Ogilvie, R. ments may be rapidly processed for ACKNOWLEDGMENTS Finniear, D. Jenner, R. Anand, J.C. DNA sequence analysis. By applying a The competent assistance of Mrs. Elsy Smith, and A. Markham. 1990. A solid-phase sequencing procedure, no Johnson is gratefully acknowledged. novel, rapid method for isolation of cloning is required and nucleotide se- Drs. David Schlessinger and Michele terminal sequences from yeast quence information can be acquired d'Urso generously provided YAC artificial chromsome (YAC) clones. for the end fragments within 48 hr of clones and information on their Nucleic Acids Res. 18: 2887-2890. the identification of a given YAC orientation. The sequence information 9. Copley, C.G., C. Boot, K. Bundell, clone. The use of fluorescent-labeled for ZFX was kindly supplied by Dr. and W.L. McPheat. 1991. Unknown primers representing sequences located David Page. Ulf Gyllensten and Marie- sequence amplification: Application 3 ' of the previously used YAC-derived Louise Steen offered valuable com- to in vitro genome walking in oligonucleotides should provide an ex- ments on the manuscript. This work Chlamydia trachomatis L2. tra level of specificity and accordingly was supported by the Beijer founda- Biotechnology 9: 74-79. should further enhance the perfor- tion, the Swedish National Board for 10. Mueiler, P. and B. Wold. 1989. In vivo mance of the procedure. Technical Development, and the Medi- footprinting of a muscle specific The CPCR protocol should be of cal Research Council. U.L. is a recipient enhancer by ligation mediated PCR. general utility in a number of situa- of a fellowship from the Wallenberg Science 246: 780-786. tions. For example, the isolation of a Foundation. 11. Pfeifer, G.P., S.D. Steigerwald, P.R. DraI-limited fragment from the 6- Mueller, B. Wold, and A.D. Riggs. globin gene, using primers derived REFERENCES 1989. Genomic sequencing and from the [~-globin gene, illustrates the 1. Saiki, R.K., D.H. Gelfand, S. Stoffel, methylation analysis by ligation possibility of applying CPCR to isolate S.J. Sharf, R. Higuchi, G.T. Horn, and mediated PCR. Science 246: 810-813. homologous gene sequences. CPCR H.A. Erlich. 1985. Enzymatic 12. Fors, L., R. Saavedra, and L. Hood. primers corresponding to a genomic amplification of [~-globin genomic 1990. 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specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517. 16. Hultman, T., S. Stahl, M. Hornes, and M. Uhl~n. 1989. Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Res. 17: 4937-4946. 17. Nakahori, Y., O. Takenaka, and Y. Nakagome. 1991. A human X-Y homologous region encodes "Amelogenin." Genomics 9: 264-269. 18. Page, D., R. Mosher, E. Simpson, E. Fisher, G. Mardon, J. Pollack, B. McGillivray, A. de la Chapelle, and G. Brown. 1987. The sex-determining region of the human Y chromosome encodes a zinc finger protein. Cell 51: 1091-1104. 19. Landegren, U., R. Kaiser, J. Sanders, and L. Hood. 1988. A ligase-mediated gene detection technique. Science 241: 1077-1080. 20. Burke, D., G. Carle, and M. Olson. 1987. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236: 806-812. 21. Dahl, N., K. Hammarstr~m-Heeroma, P. Goonewardena, C. Wadelius, K.-H. Gustavson, G. Holmgren, G.J.B. van Ommen, and U. Petterson. 1989. Isolation of DNA probe of potential use for diagnosis of the fragile X syndrome. Hum. Genet. 82: 216-218. 22. OIson, M., L. Hood, C. Cantor, and D. Botstein. 1989. A common language for physical mapping of the human genome. Science 245: 1434-1435. 23. Green, E.D. and M.V. Olson. 1990. Systematic screening of yeast artificial-chromosome libraries by use of the polymerase chain reaction. Proc. Natl. Acad. Sci. 87: 1213-1217.

Received July 22, 1991; accepted in revised form August 15, 1991.

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Capture PCR: efficient amplification of DNA fragments adjacent to a known sequence in human and YAC DNA.

M Lagerström, J Parik, H Malmgren, et al.

Genome Res. 1991 1: 111-119 Access the most recent version at doi:10.1101/gr.1.2.111

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