Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1997-05-07
1999-09-14
Degen, Nancy
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 9152, 4353201, C12P 1934, C12N 1510
Patent
active
059522011
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to the preparation of nucleic acid probes and primers as well as vectors therefor.
BACKGROUND OF THE INVENTION
The polymerase chain reaction, or PCR, provides a highly efficient method of isolating a desired gene sequence(s) from different DNA samples. One general requirement is that sequence information is available from both ends of the fragment to be amplified in order to synthesize specific amplification primers. The expense of sequencing DNA and then chemically synthesizing primers still limits the scope of many applications. In many investigations it would therefore be most helpful if the steps of DNA sequencing and oligonucleotide synthesis could be avoided entirely before genomic fragments are amplified by PCR. This is true for instance for the large programs designed to analyze the degree of polymorphism of specific DNA segments in individuals in a population, in order to identify polymorphic markers in the human or other genomes. Such genetic markers are required for mapping genetic disease and to establish or extend genetic linkage maps for a variety of organisms.
One approach which partially overcomes the above problem is the use of relatively nonspecific amplification with a limited set of primers under nonstringent conditions, so called RAPD markers. These markers serve as a means to establish relatively random polymorphic markers, analyzable by PCR without requiring DNA sequencing and chemical oligonucleotide synthesis of specific primers.
SUMMARY OF THE INVENTION
The present invention provides a different strategy to overcome the deficiences of the prior art methods and permits enzymatic synthesis of specific amplification primers, ligation probes or other probes of known structure but partially unknown sequence without access to clone specific DNA sequence information.
In one aspect, the invention provides a vector for the preparation of a nucleic acid sequence of known structure but partially unknown sequence, capable of hybridizing to a target DNA sequence without requiring knowledge of this DNA sequence, which vector comprises a site for the insertion of a DNA fragment related to the target DNA sequence, and a recognition sequence for an asymmetrically cleaving restriction enzyme or enzymes on one or both sides of said insertion site.
In another aspect, the invention provides a method of preparing a nucleic acid sequence of known structure but partially unknown sequence, capable of hybridizing to a target DNA sequence without requiring knowledge of this DNA sequence, which method comprises the steps of ligating or otherwise linking a DNA fragment related to the target sequence to a nucleotide sequence containing the recognition motif of an asymmetrically cleaving restriction enzyme, and subjecting the construct to the restriction enzyme to cleave the construct within the DNA fragment part thereof.
In still another aspect, the invention provides a method for amplifying a DNA fragment without requiring knowledge of the DNA sequence of the fragment by using enzyme-synthesized primers prepared according the above aspect of the invention.
In yet another aspect, the invention provides a method for constructing probes for target-dependent ligation reactions.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description thereof which includes an illustrative example of the practice of the invention.
The invention will be described in more detail below with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B are a schematic representation, in the form of four parts A-D, of a strategy to derive amplification primers enzymatically. Part A shows the construction of a specialized cloning vector, part B shows the cloning of an AluI fragment into the vector, part C shows enzymatic synthesis of amplification primers, and part D shows amplification of sequences corresponding to those cloned in the vector using primers derived from the ampl
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Landegren Ulf
Sundvall Mats
Degen Nancy
Larson Thomas G.
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