Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-21
2001-11-06
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06312913
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for identifying unknown nucleic acid sequences based on known nucleic acid sequences. In particular, the present invention is directed to methods for identifying coding or regulatory regions of a gene based on a known sequence.
BACKGROUND OF THE INVENTION
The polymerase chain reaction (PCR) is a method which permits the specific in vitro amplification of DNA sequences (Mullis et al.,
Cold Spring Hath. Syrup. Quant. Biol.,
51:263-273 (1986); Saiki et al.,
Science,
239:487-491 (1988); Saiki et al.,
Science,
230: 135-1354 (1985). PCR occurs by primer annealing to and extension from a template nucleic acid. Conventionally, PCR is performed using two primers that are complementary to known sequences in the template, each flanking a region of the template nucleic acid that is to be amplified. PCR is used in a wide variety of applications in a wide range of fields from molecular biology to forensics to paternity testing. Regardless of its application, the basis for any of the PCR-based assays is that a region of DNA is amplified billions fold. This makes the process of purifying a target sequence from other sequences unnecessary, as the target sequence will be the overwhelming product of the reaction.
PCR is useful for the cloning of unknown flanking cDNA sequences when part of the cDNA sequence is known. A number of methods such as rapid amplification of cDNA ends (RACE), anchored PCR, and asymmetric PCR have been used to identify flanking regions. These techniques have been used to identify sequences 3′ and 5′ of the known sequence.
A major obstacle in existing methods for the PCR amplification of specific sequences is the occurrence of nonspecific amplification products. Under PCR conditions, the stringency of the priming (Sommer and Tautz,
Nucleic Acids Res.,
17:6749 (1989)) is seldom high enough to generate a pure product longer than 1 kilobase (kb) in highly complex mixtures. This limits both the purity of the reaction product and the length of the amplifiable DNA. The use of nested primers (Haqqi et al.,
Nucleic Acids Res.,
16:11844 (1988) and size selection of the regions of interest by gel purification of the template (Ochman et al.,
Genetics,
120:621-623 (1988); Beck and Ho,
Nucleic Acids Res.,
16:9051 (1988)) diminish this problem, but high background due to insufficient stringency during the PCR amplification of genomic DNA remains a significant problem. Methods to amplify unknown flanking DNA using nested PCR or with gel purified template still result in limited specificity, as the initial PCR amplification using these methods does not improve upon the specificity level conferred by conventional two primer PCR.
Many PCR techniques have been developed for sequencing DNA fragments flanking known sequences. Of them, inverse PCR is a matter of interest because of the simplicity of its principle. (Huang S H.,
Methods Mol Biol.
69:89-96 (1997)). However, the protocols for inverse PCR introduced to date use time-consuming procedures, and the methods are limited by the number of suitable restriction enzymes and the presence of restriction sites in the DNA of interest.
Another method that has permitted the highly specific amplification of >2 kb of unknown DNA that flanks a known sequence from bulk human genomic DNA is panhandle PCR (Jones and Winistorfer, Nucleic Acids Res., 20:595-600 (1992); Jones and Winistorfer,
PCR Methods Applic.,
2:197-203 (1993)). This method involves primer-dependent attachment of a known sequence to the uncharacterized flanking region of a specific DNA strand which contains an unknown sequence. Generation of the panhandle template permits PCR amplification of the unknown DNA because known sequence now flanks the unknown DNA in those strands that contain the unknown DNA. However, in the panhandle PCR method, the initial priming during the amplification reaction must compete with intra-strand annealing of a long inverted repeat that comprises the handle of the panhandle template, which diminishes the efficiency of this necessary first step.
There is thus a need in the art for a method of identifying sequences both 5′ and 3′ of known sequences, which method provides greater specificity and less contamination products from the genomic sequences.
SUMMARY OF THE INVENTION
The present invention provides methods for isolating polynucleotides flanking a known nucleic acid sequences and reagents for using such methods.
One embodiment of the invention provides a method comprising the steps of 1) attaching a linker to the ends of a nucleic acid which has both known and unknown nucleic acid sequences; 2) attaching an adaptor to the nucleic acid via the linkers to form a circularized nucleic acid; and 3) performing an amplification reaction using the circularized nucleic acid as a template, with one primer specific to the known nucleic acid sequence, and a second primer specific to the adaptor. The reaction is designed to produce a product having unknown sequences of the nucleic acid. The product can then be sequenced to determine the unknown sequences. This method is illustrated in FIG.
1
.
Another embodiment of the invention provides a method having the steps of 1) attaching a linker to the ends of a nucleic acid; 2) attaching an adaptor to the nucleic acid via the linker to form a circularized nucleic acid; 3) performing an first amplification reaction using the circularized nucleic acid as a template and two primers specific to the known region of the nucleic acid to obtain a product; and 4) performing a second amplification reaction using the product as a template, with one primer specific to the known nucleic acid sequence, and a second primer specific to the adaptor. The linker preferably provides ends that are compatible for the binding and ligation of the adaptor, but are incompatible for self-ligation of the nucleic acid without the adaptor. The adaptor allows for increased accuracy of the first amplification step, resulting in fewer amplification artifacts and higher efficiency of subsequent amplifications. The second amplification product provides a selection step to identify the products that are specific to the molecules having an adaptor, thus enriching the population of amplification products to increase the number of products that provide the sequence of the nucleic acid flanking the portion having a known sequence. The method is illustrated in FIG.
2
.
In another embodiment, the invention provides a method for performing simultaneous amplification reactions on multiple circularized nucleic acids. In this embodiment, the primers used to amplify each circularized nucleic acid are preferably designed to allow maximum efficiency for the amplification reaction of each sample. For example, primers can be designed to have similar T
m
s, e.g., by determination of G-C content of the primers, and are preferably designed to avoid secondary structure of the primer. The design of the primers can also be automated using a computer program which can identify appropriate primers for use in the present invention.
A feature of the invention is that the first amplification product can be further selected prior to the second amplification reaction.
Another feature of the invention is that additional amplifications may be performed following the second amplification to firther select for a desired final product.
Yet another feature of the invention is that primers to multiple nucleic acids can be designed using an automated selection process, e.g., a computer program.
An advantage of the present invention is that fewer nonspecific products are produced, particularly in the first amplification reaction.
Another advantage of the methods of the invention is that they may be used to isolate and characterize sequences 3′ and/or 5′ of a known sequence of a nucleic acid.
Yet another advantage of the present invention is that sequences 3′ and 5′ of the known sequences can be determined using a single circularized template.
Yet another advantage
Fu Glenn K.
Lee Walter H.
Stuve Laura L.
Wang Jonathan T.
Borden Paula
Bozicevic Field & Francis LLP
Campbell Eggerton A.
Chunduru Prabha
Incyte Genomics Inc.
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