Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking

Several PCR-based methods are available for walking from a known region to an unknown region in cloned or uncloned genomic DNA. The methods are of three types: inverse PCR (1), randomly primed PCR (2) and adaptor ligation PCR (3-6). However, these methods have not been generally applied to walking in uncloned genomic DNA because they are either complicated or inefficient. Recent improvements to these methods have been applied to uncloned genomic DNA (7,8), however, walks have been limited to distances of <1 kb. We have investigated the application of 'long and accurate PCR' (9,10) to walking in uncloned genomic DNA. We initially examined the use of 'unpredictably primed PCR' (3), a new method based on randomly primed PCR and 'vectorette PCR' (5), which is based upon adaptor ligation. As a model system we attempted to walk upstream from exon 1 of the human tissue-type plasminogen activator (TPA) gene which has been previously characterized (11). In our hands both 'unpredictably primed PCR' and 'vectorette PCR' generated multiple PCR products even after nested PCR was perforned. Although upon analysis some of the PCR products were found to be derived from successful walks, the presence of multiple PCR products would complicate further characterization without Southern blot hybridization or extensive cloning. We have improved upon the adaptor ligation method by combining 'vectorette PCR' with a newly developed method termed 'suppression PCR' (12). A special adaptor is ligated to the ends of DNA fragments generated by digestion of human genomicDNA with EcoRV, Scal, Dral, PvuII and SspI separately. Following adaptor ligation, a small amount of the DNA is used

An improved PCR method for walking in uncloned genomic DNA

The invention relates to a process for the controlled amplification of at least one part of a starting DNA containing a plurality of restriction sites for a determined specific restriction endonuclease, and of which at least part of its nucleic acid is unknown. Application of this process to human, animal or plant DNA fingerprinting, to identification of restriction fragment length polymorphisms. Kit for the application of the process.

Selective restriction fragment amplification : a general method for DNA fingerprinting

Isolation of unknown DNA sequences flanked by known sequences is an important task in molecular biology research. Thermal asymmetric interlaced PCR (TAIL-PCR) is an effective method for this purpose. However, the success rate of the original TAIL-PCR needs to be increased, and it is more desirable to obtain target products with larger sizes. Here we present a substantially improved TAIL-PCR procedure with special primer design and optimized thermal conditions. This high-efficiency TAIL-PCR (hiTAIL-PCR) combines the advantages of the TAIL-cycling and suppression-PCR, thus it can block the amplification of nontarget products and suppress small target ones, but allow efficient amplification of large target sequences. Using this method, we isolated genomic flanking sequences of T-DNA insertions from transgenic rice lines. In our tests, the success rates of the reactions were higher than 90%, and in most cases the obtained major products had sizes of 1–3 kb.

High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences

Preparation and application of new monosized polymer particles

Recently, a method named inverse PCR has been described by which genomic DNA sequences adjacent to a known (sequenced) locus can be amplified in vitro and sequenced (1-2). The method comprises the following three steps: (i) restriction of genomic DNA; (ii) recirculanzation of linearised fragments by ligation; and (iii) in vitro amplification with two primers which are reversed in their direction in respect to their normal orientation for PCR. The circularization reaction is unreliable and often concatamers are observed. We have developed a modified PCR method which alows walking and sequencing in any direction along genomic DNA starting from a known sequence. The basic principle of the method is outlined in figure la. There are several advantages of the new method. Ligation of ohgo-cassettes with sticky ends does not cause any problem. Since the cassette contains the universal primer sequence sequencing of PCR products can be done with any of the existing dideoxy termination or chemical degradation techniques using radioor fluorescently-labelled nucleotides or primers. PCR products can easily be sequenced from both ends. The sequence obtained from the specific primer confirms the overlap and the sequence from the universal primer gives the necessary information from the endpoint of the PCR product to design new primers for the next walking/sequencing step. We have successfully walked along the nematode unc 31 gene contained on a YAC within the background of total yeast DNA (figure lb). The technique has also been used to walk along total human DNA from exon 51 of the DMD gene into the adjacent intron. Each round of oligo-cassette mediated PCR walking was performed using multiple restriction enzymes (EcoRI, HindlTJ, Xbal, BglTI, Hinfl). The method can be applied in combination with any restriction endonuclease. Protocol: ds oligo-cassettes were 28 nucleotides long having a 4 nt or 3 (Hinfl) nt overhang. Genomic DNA (250-500 ng) was digested to completion and the restriction enzyme inactivated by heating. Half of the DNA was ligated to 5-50 pmol of oligocassette in a total of 20 y\\. Following dilution to 100 /xl and heat inactivation 1 /d of the ligated product was used for the first round linear PCR with a specific primer which had been biotinylated at its 5'-end. PCR was carried out in 1 xPCR buffer (Cetus), 250 tiM dNTP's, 0.5 /tM specific biotinylated primer and 2.5 units Taq polymerase (Cetus). Since only one primer is present the amplification (50 cycles of 95°C 0.5 mm, 55°C 1 min, 72°C 1 min) is linear and not exponential. Isolation of biotin-labelled products was performed using 25 /xl of washed streptavidin-coated beads (Dynal S.A.). After incubation for 15 min at RT the beads were washed three times with 40 yl of 1 M NaCl in TE buffer followed by three washes with 1 xTE buffer. Supernatants were carefully removed after each washing. Exponential PCR was earned out using the polymer bound DNA as the template as described above except that 35 cycles were carried out and two primers were present, (one complementary to the cassette and the other unbiotinylated but specific to the known sequence). Amplification products were analysed on 1% agarose. After reamplification, specific PCR products were separated on 1 %

A. Rosenthal

Papers

Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction.