Chromosome Walking by Inverse PCR

21 Chromosome Walking by Inverse PCR Michael Gotesman, Selwyn A. Williams, Jorge A. Garcés, and Ray H. Gavin CONTENTS 21.1 Introduction................
Author: Myra Chase
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Chromosome Walking by Inverse PCR Michael Gotesman, Selwyn A. Williams, Jorge A. Garcés, and Ray H. Gavin

CONTENTS 21.1 Introduction........................................................................................................................... 299 21.2 Materials and Methods.......................................................................................................... 301 21.2.1 Primers....................................................................................................................... 301 21.2.2 DNA Templates......................................................................................................... 301 21.2.3 Circularizing DNA Templates...................................................................................302 21.2.4 Inverse PCR...............................................................................................................302 21.2.5 Analysis of PCR Products.........................................................................................302 21.2.6 Cloning and Sequencing the PCR Products..............................................................302 21.2.7 Chromosome Walking Using Sequencing Results.................................................... 303 21.3 Results.................................................................................................................................... 303 21.4 Discussion.............................................................................................................................. 303 Acknowledgment............................................................................................................................304 References.......................................................................................................................................304

21.1 INTRODUCTION Inverse polymerase chain reaction (iPCR) is a powerful tool that can be used for the sequence analysis of DNA when only one end of a DNA sequence is known. iPCR1–3 amplifies DNA from a circular template using primers with their 3′ ends directed away from each other (Figure 21.1). Cloning and sequencing PCR products are facilitated by the use of vectors, for example, pCR®2.1 (Invitrogen), which contain a thymine overhang. iPCR is very useful for supplementing conventional PCR screens that often yield only a substantial fragment of the gene of interest. An iPCR strategy used in conjunction with commercial cloning vectors and modern sequencing technology can rapidly yield accurate DNA sequencing results for several kilobases (kB) and allows one to “walk” both upstream and downstream of the known DNA sequence to obtain additional sequence. The methodology facilitates amplification of unknown DNA flanking sequences without the labor involved in constructing and screening libraries. Identification of flanking sequences on either side of a known sequence can be challenging. Other PCR-based methods for gene isolation, including random priming or the use of adapter sequences (anchored and rapid amplification of cDNA ends (RACE) PCR), employ a single sequence-specific primer, consensus or degenerate primer, or randomly prime off the template. These approaches frequently yield a low signal-to-noise ratio. iPCR requires two sequence-specific primers and generally yields a high signal-to-noise ratio. In addition, the iPCR primer pair can be used to amplify multiple circularized gene fragments that contain overlapping restriction sites. However, the iPCR technique requires circularization of DNA fragments within an optimum size range (see Section 21.4). iPCR has been useful in studies of genomic DNA from a variety of models, including humans,1–3 Lactobacillus,4 the red seaweed Grateloupia,5 Vibrio,6 Tetrahymena,7–9 the marine yeast Williopsis 299

300

PCR Technology Region of interest Known sequence

EcoRI

EcoRI

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EcoRI

HindIII

HindIII

HindIII

Hind III

EcoRI

EcoRI

HindIII HindIII

EcoRI HindIII HindII

FIGURE 21.1  (See color insert.) Diagram of the inverse PCR strategy for chromosome walking.

saturnus,10 and environmental metagenomes.11 iPCR is amenable to studies of protein analysis,12–14 DNA regulatory sequences,15,16 RNAi,17 and insertional mutagenesis.18–21 Other recent use of this technique includes the detection of chromosome rearrangement in cancer patients,22 sex determination in Bubalus bubalis (cattle),23 and locating sites of integration in mice using Cre/loxp recombination system24 and in silkworm using the piggyBac transposon system.25 In this chapter, we describe protocols for using chromosome walking with iPCR to clone and sequence contiguous DNA upstream and downstream of a known site.

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21.2  MATERIALS AND METHODS The main steps in iPCR (Figure 21.1) are as follows:

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1. Purify and cut the genomic DNA sample with an appropriate restriction enzyme (EcoRI or HindIII in this example). 2. Ligate the fragments into circular templates. 3. Perform iPCRs using primer sequences within the region of known sequence. 4. Analyze iPCR products with appropriate restriction endonucleases (EcoRI and HindIII in this example). 5. Ligate iPCR product into pCR2.1 and use the ligated vector to transform Escherichia coli. 6. Use blue/white screening to search for candidate screening colonies. 7. (Optional) Reanalyze plasmid with appropriate restriction endonucleases (EcoRI and HindIII in this example). 8. Use M13F and M13R to sequence plasmids. 9. After the iPCR product has been cloned and sequenced, a full-length contiguous fragment may be amplified by conventional PCR using primer sequences at or adjacent to the restriction sites from a genomic DNA template. 10. Use sequencing results to search for new primers 5′ and 3′ of the starting fragment to yield more sequence of interest. 11. Cut the genomic DNA sample with the second restriction enzyme. This creates additional fragments that will serve as template for the iPCR reaction and facilitates amplification of novel sequences both upstream and downstream of the known sequence (Figure 21.1; second set of HindIII sites and primers denoted by red arrows). 12. Obtain additional clones by iPCR. The overlap between the new and old clones allows for proper orientation of the iPCR fragments and prevents the creation of gaps in a contiguous sequence. PCR primers are designed from regions of known sequence as depicted in Figure 21.1. 13. Amplify full-length contiguous DNA fragment by conventional PCR using primer sequences at or adjacent to the outermost restriction sites. Using this strategy, between 5 and 10 kb of novel contiguous DNA sequence information can be obtained after only two rounds of iPCR. Further iPCR cycles using fragments left and right of the starting fragment will yield more sequence. Each cycle of the strategy allows the user to “walk” both upstream and downstream of a known DNA sequence.

21.2.1  Primers We have used a variety of primers for the amplification of ciliated protozoan sequences. The primers were designed to amplify from circular templates and therefore have their 3′ ends directed away from each other. These primers were generally 21- to 24-mers and 50% GC-rich with a Tm between 60°C and 68°C. A stock solution at 100 µM was prepared by resuspending the primers in sterile distilled water or TE buffer (1×).

21.2.2  DNA Templates Tetrahymena genomic DNA was prepared by using a commercially available DNA Extraction Kit (Stratagene) with a final ethanol precipitation step. DNA was digested in small tubes, each containing 30 µg of genomic DNA, 30 units of EcoRI or HindIII, enzyme buffer (supplier’s directions), and distilled water to 50 µL. The tubes were incubated at 37°C for 1 h. Restriction digest products were purified using a Chromospin column (Clontech). Alternatively, DNA digestion products were purified by gel electrophoresis, which was also useful for the identification of components of optimum size for circularization (see Section 21.4). For gel purification, the band of interest was excised with a clean, sharp razor blade and purified using a Gel Extraction Kit (Qiagen).

302

PCR Technology

21.2.3  Circularizing DNA Templates

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Serial dilutions of the digested, purified DNA were prepared. A range of ligation reactions with varying concentrations (10 ng to 1.0 μg) of the purified DNA digests was set up in small tubes. Each reaction mixture contained the appropriate amount of diluted DNA and six Weiss units of T4 ligase in ligation buffer consisting of 66 mM Tris–HCl (pH 7.6), 6.6 mM MgCl2, 0.1 mM ATP, 0.1 mM spermidine, 10 mM DTT, and stabilizers. The tubes were incubated at 16°C for 60 min. The circularized fragments were purified using a CHROMASPIN column.

21.2.4 Inverse PCR Serial dilutions of the circularized DNA described in Section 21.2.3 were prepared, and PCRs were set up using varying concentrations of circularized DNA. Each PCR contained • • • • • •

5 µL 10× Advantage 2 PCR1 Buffer (Clontech Laboratories)* 1 µL 50× dNTP mix† 1 µL (each) primers (0.5 µM each) 5 ng to 0.5 µg circularized DNA 1 µL 50× Advantage 2 Polymerase Mix3 (Clontech Laboratories)‡ PCR Grade water to 50 µL

The PCR program consisted of • • • • •

Pre-PCR hold at 94°C for 10–15 s 18 rounds of denaturation at 94°C for 12 s Annealing/extension at 65°C for 3–5 min (1 min/kb) 12 cycles of denaturation at 94°C for 12 s Annealing/extension at 65°C for 3–5 min (1 min/kb, adding time increments of 12 s per cycle) • Final extension at 72°C for 10–15 min to add 3′ “A” overhangs

21.2.5  Analysis of PCR Products PCR products were analyzed on 1% agarose gels in Tris-acetate-ethylenediaminetetraacetic acid (TAE) buffer using standard electrophoresis protocols. To confirm that the PCR product was the result of authentic amplification from a circular template, an aliquot of the PCR product was digested with the same enzyme (EcoRI or HindIII) that generated the original template DNA, and the digestion products were analyzed by gel electrophoresis.

21.2.6  Cloning and Sequencing the PCR Products The PCR-amplified products were cloned into pCR2.1 by ligating 3 μL of PCR product to 1 μL of pCR2.1 in a solution that consisted of 1 μL of T4 ligase, 1 μL of 10× ligase buffer, and 4 μL of sterile water incubated overnight at 4°C. The ligation product was used to transform E. coli that were subsequently plated on Luria-Bertani agar supplemented with 100 μg/mL ampicillin and X-Gal. 10× Advantage 2 PCR Buffer contains: 400 mM Tricine–KOH (pH 8.7 at 25°C), 150 mM KOAc, 35 mM Mg(OAc)2, 37.5 µg BSA, 0.05% Tween-20, 0.05% Nonidet-P40. † 50× dNTP mix contains: 10 mM each of dATP, dCTP, dGTP, and dTTP, final concentration of 0.2 mM each nucleotide. ‡ 50× Advantage 2 Polymerase Mix contains Titanium Taq DNA polymerase, a nuclease-deficient, N-terminal deletion of Taq DNA polymerase plus TaqStart Antibody to provide automatic hotstart PCR, and a minor amount of a proofreading polymerase. *

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Plasmids were extracted from white E. coli colonies and reanalyzed on 1% agarose gels as previously described. M13F and M13R primers were used to sequence the plasmid.

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21.2.7  Chromosome Walking Using Sequencing Results Sequencing results can be used to design new primers 3′ and 5′ of the original primer site for repeated rounds of iPCR. Typical DNA sequencing reactions yield about 1000 bases; therefore, new primers that are about 100 bases from the end of original sequencing results should be used to extend/walk up the gene.

21.3 RESULTS Circularized EcoRI- or HindIII-generated genomic DNA fragments yielded sequence information upstream and downstream of the original known DNA sequence (Figure 21.1). PCR amplification of sequences in circularized EcoRI-digested genomic DNA yielded sequences predominantly downstream of the PCR priming site (Figure 21.1). Amplification of circularized HindIII-digested fragments produced sequences upstream and downstream of the PCR priming site (Figure 21.1). Digestion of an aliquot of the amplified product with either EcoRI or HindIII produced two fragments as expected for authentic amplification from a circular template (Figure 21.1).

21.4 DISCUSSION In this chapter, we have described a general method that can be used to apply the iPCR technique to clone contiguous sequences upstream and downstream of a known DNA sequence. Although the technique has been described for the amplification of Tetrahymena sequences, it is readily adaptable for use with any genomic DNA template. A suitable restriction enzyme should generate fragments that are 2–3 kB in length and contain a four base overhang to facilitate ligation. Select an enzyme that has a recognition site predicted to occur frequently within the template DNA based on GC and AT content. For example, in Tetrahymena, the AT content of genomic DNA is high, and enzymes such as EcoRI and HindIII, which cut at AT-rich sites, yield fragments in the desired size range. In contrast, enzymes such as BamH1, which cut at GC-rich sites, yield fragments that are too long for efficient use in iPCR. Hybridization blotting can be used to confirm the identity of the restriction fragments. The Tm of the primers should not differ by more than 3–5°C from each other. If the Tm of the primers varies by 5–10°C, use a two-step PCR from the highest to the lowest Tm value. For example, in a two-step PCR reaction in which the Tm of one primer is 68°C and the Tm of the second primer is 62°C start with a cycle consisting of a 94° denaturation step followed by a 68°C combined annealing/extension step. Subtract 0.2°C from the annealing/extension step in each subsequent PCR cycle. A 5-s time increment should be added to the annealing/extension step of each subsequent cycle to compensate for a slight decrease in the DNA polymerase’s rate of nucleotide incorporation as the reaction progresses. The last of 30 cycles consist of a 94°C denaturation step followed by a 62°C combined annealing/extension step. Taq polymerase is ideal for the iPCR technique described in this chapter. Taq polymerase can be induced to add an extra adenine 3′ overhang to replicated DNA transcripts and therefore can be used to clone the transcript directly into pCR2.1 or other commercial cloning vectors that are prelinearized with 3′ thymine overhangs. The multiple cloning site of pCR2.1 is in the lacZ gene, and supplementation of media with X-Gal helps researchers use blue/white screening to locate plasmids containing inserts. Cloning a DNA transcript into pCR2.1 allows use of M13R and M13F primer sites within pCR2.1 to sequence the vector insert and circumvents the need to create new sequencing primers.

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PCR Technology

Many of the problems encountered with iPCR can often be traced to characteristics of the template DNA. In creating the circular DNA template, there is competition between concatamer formation and circularization of DNA fragments. The optimum DNA concentration that promotes circularization varies with the length of the DNA to be circularized and must be determined empirically for each template. In general, the optimum size range for efficient circularization is between 2 and 3 kb. Fragments outside this range fail to circularize efficiently. To lower the high melting temperatures required for denaturation of closed circular DNA, the DNA can be linearized at a site between primers. Alternatively, a PCR buffer containing dimethyl sulfoxide (DMSO) and glycerol (5%) as cosolvents can be used to overcome difficult secondary structure restrictions. Although iPCR products are derived from monomeric circular templates, the presence of concatamers, even at low DNA concentrations, can yield unwanted amplification of noncontiguous sequences. A quick and effective way of distinguishing between an iPCR product and a concatamerbased amplification is to digest an aliquot of the amplified product with the same restriction enzyme used to generate the genomic template fragments. An authentic iPCR product contains a single internal restriction site corresponding to the original template-generating enzyme. Restriction digestion using this enzyme would therefore cleave the PCR product into two subfragments that, in most cases, are readily resolvable by agarose gel electrophoresis. However, if the restriction site is equidistant from both primers, both subfragments will have the same molecular size. The sum of the molecular sizes of the two subfragments should be equal to the size of the original iPCR product. A restriction digest of a concatamer-based amplification will always yield in excess of two subfragments due to the presence of multiple internal cleavage sites. If the iPCR generates multiple amplification products, each band should be excised and gel-purified separately. Aliquots of the gel purified DNA can then be used for restriction digestion analysis. This method provides a simple means of verifying an iPCR product prior to cloning and sequencing. Amplify3.1 (http:// engels.genetics.wisc.edu/amplify/) is a helpful algorithm in designing primers for amplification by quickly simulating PCRs, thereby helping researchers design primers that are least likely to target undesired regions.

ACKNOWLEDGMENT The original research on the use of inverse PCR with Tetrahymena DNA was supported by grants MCB 9808301, MCB 0130624, and 0517083 from National Science Foundation to R.H.G.

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