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Optimization of Long-Distance PCR Using a Transposon-Based Model System

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Optimization of Long-Distance PCR Using a Transposon-Based Model System Downloaded from genome.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Optimization of Long-distance PCR Using a Transposon-based Model System Lynne D. Ohler and Elise A. Rose I Human Genome Laboratory, Perkin-Elmer Cetus Instruments, Emeryville, California 94608 The ability to amplify routinely long starts ''(7) and, lastly, the use of auto- and cost involved in laboratory research. PCR products (5-25 kb) with high segment extension thermocycling. In addition, focus will shift to analysis of specificity and fidelity, regardless of These results also provide Insights genomic regions that have been recalci- target template sequence or struc- into additional approaches that trant to current mapping and sequenc- ture, would provide significant might further enhance our ability to ing approaches. Such regions include benefits to genome mapping and perform long-distance PCR. those that are especially rich in guanine sequencing endeavors. Although oc- (G) and cytosine (C), or contain complex casional reports have described the secondary structures. Efficient amplifica- generation of long PCR prod- tion through these regions would facili- ucts, (1-4) such results have been dif- The versatility and power of the poly- tate analysis and characterization of ficult to replicate and have fre- merase chain reaction (PCR) has encour- many of the gaps in current genomic quently utilized probe hybridization aged its involvement in almost every maps. to Identify the specific product from aspect of genome mapping and sequenc- The ability to amplify routinely tem- nonspecific amplified DNA. Produc- ing. (8) The application of PCR has been plates as large as 5-25 kb, regardless of tion of specific PCR products has gen- central in formulating both the concep- DNA sequence or structure, would repre- erally been limited to target tem- tual and practical approaches on which sent a major breakthrough in human ge- plates of less than 3 kb. (s) To extend the entire Human Genome Project is nome mapping research. Such "long- the effective range of standard PCR based, as evidenced by the adoption of range PCR" would maintain continuity, amplification, it may be necessary to the sequence tagged sites (STS) pro- order, and orientation of DNA fragments utilize alternative reaction condi- posal. ~9) Other genomic mapping appli- and would provide longer templates tions and/or components, such as cations of PCR technology include its for subsequent sequencing endeavors. novel thermostable DNA polymerases use in isolation of human DNA frag- Moreover, reliable amplification over or accessory proteins. We describe ments present in hybrid cell lines or long distances would decrease the num- the use of a model system to eval- from microdissected or flow-sorted chro- ber of PCR amplifications that would uate systematically methodological mosomal regions~m); chromosomal need to be performed to analyze any changes that might enable efficient walking and expansion of contiguous given genomic region, eliminating the long-range PCR. Specifically, the yeast artificial chromosome (YAC), need for additional primers and other re- transposon TnSsupF has been used cosmid, or phage clones~11'12); finger- agents, and thus decreasing both the to Introduce randomly identical, printing of DNA fragments for ordering time and cost involved in genome map- known primer binding sites within and orienting YAC, cosmid, or phage ping efforts. Long-range PCR would also separate Isolates of phage clones car- clones~m); fingerprinting of DNA in hy- greatly reduce the number of overlap- rying Identical Inserts. (6) Transpo- brid cell lines for identification (and to ping regions that would need to be com- son-based PCR allows us to study am- check for stability of human DNA con- pared, as well as the need for extensive plification of DNA fragments that tent)~m); mapping and analysis of ex- subcloning. vary in size and sequence using only a pressed sequences from a particular Current limitations in the size of PCR single set of primers, in the present genomic region~13~; and direct PCR-cou- products that can be generated reliably studies, we describe conditions that pied DNA sequencing. ~14'1s) are on the order of 3-4 kb. (s) Although enable PCR amplification of specific As efforts to map and sequence com- occasional reports have appeared de- DNA templates ranging in size up to plex genomes continue, there is an ever- scribing detection of large PCR prod- 9 kb. Some of the key features of increasing need to develop streamlined ucts/1-4) little effort has been made to our methodology Include the use of methodologies that will reduce the time optimize conditions for long-distance recombinant Thermus thermophilus PCR. As a consequence, such results have (rTth) DNA polymerase, the addition been difficult to replicate and frequently 1Present address: Advanced Center for Genetic Tech- of gelatin to the reaction mixture, nology, Applied Biosystems Inc., 850 Lincoln Centre required probe hybridization to identify the use of wax-mediated "hot Drive, Foster City, CA 94404. the specific product from the nonspe- 2:51-599 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/92 $3.00 PCR Methods and Applications 51 Downloaded from genome.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press cific amplified DNA. Conditions have yet were kindly provided by D. Berg (Wash- near each transposon end and with to be described that enable reliable am- ington University, St. Louis). A single primers specific for the two ends of the plification with high specificity and fi- Tn5supF transposon was randomly in- vector. The primers and their sequences delity of long PCR products, regardless of serted at a unique location within each are as follows: k left arm cloning site the target template sequence. cloned fragment. ~4'6~ Phage templates (L), 5'-ATAGAGTCTTGCAGACAAACTG- Our long-term goal is to explore a va- designated 4, 15, S lac, 3 lac, 2, 14, and 9 C-3'; k right arm cloning site (R), 5'-GC- riety of thermostable DNA polymerases carried transposon insertions at the sites CTAACGATCATATACATGG-3'; Tn5supF under different reaction conditions that indicated in Figure 1. The bacterial inside end (I), 5'-TAGGATCCCGAGATC- might enable (1) PCR amplification of strains MC1061 or LE392 were used in- TGATC-3'; and Tn5supF outside end longer products (5-25 kb) with high terchangably for plating of h phage. (O), 5'-TAGGATCCCCTACTTGTGTA-3'. specificity and fidelity, and (2) PCR am- Plaque isolates were dissolved in 50 txl of Primers I and O were designed by Phad- plification from regions that are cur- SM buffer (16~ and stored at 4~ Aliquots nis et al. (6~ Oligonucleotides L and R rently difficult to amplify (due to either of 5 Ixl were used for PCR amplifications. were designed using annealing thermo- complex secondary structure and/or dynamics software developed by D. high G + C content). Our model system Birch, Q. Chou, and W. Bloch (Cetus employs transposons to introduce Corp). Use of primers with similar melt- PCR Primers unique known primer binding sites into ing temperature (Tm) proved to be criti- any targeted region of DNA, thus making PCR was performed using primers that cal in obtaining specific amplification of the region amenable to PCR amplifica- are complementary to unique sequences desired long PCR products. tion and subsequent sequencing.(6~ Transposons are discrete DNA segments that move (or transpose) relatively ran- domly from one DNA site to another in the absence of apparent DNA homology. Transposon-based PCR represents a po- tential method for inserting PCR primer binding sites into regions that are diffi- cult to analyze and for which no known sequence information is available. In the current studies, the transposon Tn5supF has been used to introduce PCR primer binding sites at random locations within separate isolates of phage k 138 clones carrying identical Escherichia coli inserts. ~4'6~ PCR-mediated mapping of transposon insertions has been carried out using primers specific for TN5supF inside and outside ends in relation to the left and right h cloning sites. Using this system, only two sets of primers are re- quired to study amplification of many DNA fragments that vary in size and se- quence. Our use of Tn5supF transposon- based PCR as a model system has en- abled us to examine a variety of new reaction components and conditions, with primer binding sites spaced at in- creasing distances, without having to change PCR primers. This approach has already enabled us to define conditions that extend the range of PCR to 9 kb. MATERIALS AND METHODS DNA Templates FIGURE 1 Mapping of transposon insertions. The model system used in these studies employs DNA templates used in these studies introduction of the transposon Tn5supF into random locations of individual phage k 138 clones. were h phage 138 clones carrying the E. Each phage clone carries the E. coli LacZ gene with a small amount of surrounding DNA.(6~ coli lacZ gene with a small amount of Mapping of transposon insertions is carried out using primers specific for Tn5supF inside (I) and surrounding E. coli DNA. These clones outside (O) ends and for k DNA adjacent to the left (L) and right (R) cloning sites.(6~ 52 PCR Methods and Applications Downloaded from genome.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press PCR Amplification Reaction These reagents included Tween 20 cler 480 (Perkin-Elmer Cetus) and the Components (0.001-1.0%) and gelatin (0.005-0.05%). GeneAmp PCR System 9600 (Perkin- Elmer Cetus). Optimization of reaction PCR amplification reactions were per- Hot Start Technique conditions, however, was carried out us- formed in 50-1~1 total volumes. Each re- ing the System 9600 exclusively. For action mixture contained 5 ~l of sample The specificity of PCR amplification can these experiments, PCR amplification re- DNA template 250 ~I,M each dNTP (Per- be greatly enhanced by minimizing action mixtures were prepared in thin- kin-Elmer Cetus), and 30 pmoles each of primer extension at misprimed target walled MicroAmp reaction tubes (Per- two primers (either L or R, along with sites and reducing the rate at which kin-Elmer Cetus).
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