A Versatile Method for Accelerated Chromosome Walking and DNA Fingerprinting

Summary Background: A variety of DNA amplification methods deal with sequence analysis of an uncharacterized region adjacent to a known element. These methods include inverse PCR (iPCR), panhandle PCR, cassette ligation-anchored, and Thermal Asymmetric Interlaced PCR. Despite various degrees of success, a number of drawbacks reduce the accuracy and efficiency of each of these methods. Unsequenceable domains are frequently poor in restriction enzyme sites or contain heterochromatic DNA, which has few genes and many repeated regions that are difficult to maintain as clones for DNA sequencing. As a consequence, there is a need to develop new ways to determine these hard-to-sequence regions of nucleic acid.

Invention: A novel method for amplifying or recovering an unknown nucleic acid sequence adjacent to a known nucleic acid sequence. The method includes the following steps: -Polymerase-catalyzed extension from a known region into an unknown region by a primer-directed synthesis of a first strand -Enzymatic destruction of the first primer, typically using the single-strand-specific enzyme exonucleaseI (exoI)
-Strand denaturation and annealing and short extension of a sequence-tagged, random-ended primer across the first strand -Destruction of the second primer and repair of the branched ends in the intermediate products, such that both events are simultaneously achieved by reintroduction of an exonuclease, e.g., exoI, an enzyme which digests single-stranded DNA from the 3' end, causing removal of free primer and trimming of branched DNA back to the branchpoint Polymerase-catalyzed sequence conversion of the repaired ends that results in a complement to the specific sequence tag -Strand denaturation and formation of a lariat or stem-loop or panhandle structure by intrastrand annealing between the tag complement and a copy of the tag at the other end of the strand -Self-primed extension of the lariat; and -Polymerase amplification to generate specific final products.

The amplicons are also suitable for standard sequencing, without requiring molecular cloning; it is therefore possible to directly genome walk over a substantial distance, such that the range of the walk in this method is limited only by the capability of the polymerase component that is selected. The high performance of long-distance polymerases indicates that the current range of the UFW approaches may exceed 50 kilobases per walk. The final products, or amplicons, can also be subjected to agarose gel electrophoresis. The gel pattern, or size pattern, yields a DNA fingerprint that is particular to a combination of the flanking region's polymerase pause sites and its preferential binding sites for the second primer of this method.

Applications Advantages: One of the most significant aspects of UFW is that the method reliably generates data over stretches of DNA that are difficult to sequence or are unsequenceable by conventional methods. UFW also overcomes several drawbacks of earlier genome walking protocols. Previous methods required the use of restriction enzymes, presence of convenient restriction enzyme cleavage sites in the target DNA and use of DNA ligase. The present method is distinguished from earlier methods in that the method is carried out in the absence of a restriction enzyme and of a DNA ligase. The unknown sequence of a target nucleic acid need not contain a restriction enzyme cleavage site, and the method generates sequence information for unknown sequence ranging in size from 0.5-100 kilobases.

UFW has several other advantages over known methods. For example, UFW is performed in a single-buffer system, and reagents are directly added to a single buffer mixture in a single vessel such as a test tube or well of a microtiter plate. Volumes are typically in the microliter-scale volumes, but may be scaled up proportionately without sacrificing efficiency or accuracy. UFW is performed with reactions in multitube-, multiwell-, or microplate arrays, with miniaturization to submicroliter volumes, or with other spatial economizing. An automated or semi-automated system is used to direct the amplification reactions. UFW can be automated using a robotic workstation and a multi-well reaction chamber format.

Applications: UFW is particularly suitable for analyzing transposon sequences and sequences flanking the insertion site of a known transposon or viral element. Alternatively, the known element is a pathogen, or a natural or constructed gene or chromosome. Besides direct genome walking, other uses of UFW include high throughput mapping of genome-wide insertional mutagenesis for functional genomics, identifying vector integration sites for gene therapy studies, and tracking of viral replication by detecting the insertional activity that accompanies productive infection, as with retroviruses. The manipulations for running UFW are conducive to automation. The target nucleic acid is RNA, DNA, or cDNA. Thus, another advantage is that the method can utilize RNA as a working material. Earlier methods which require restriction enzyme digestion cannot utilize RNA because it is not cut by restriction endonucleases.

Patents: US Patent 6,929,914 For Further Information Please Contact the Director of Business Development Laura Brass Email: laura_brass@harvard.edu Telephone: (617) 495-3067

Inventor(s): Myrick, Kyl V.

Type of Offer: Licensing



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