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Selective restriction fragment amplification: fingerprinting

Inactive Publication Date: 2010-10-21
KEYGENE NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014]The invention is based on the use of a novel application of polymerase chain reaction technique (PCR) for amplifying one or more restriction fragments from complex mixtures of DNA fragments obtained by digesting genomic DNA molecules with restriction endonucleases. One particular advantage of the invention is to enable the amplification of DNA restriction fragments in situations where the nucleotide sequence of the ends of the restriction fragments are not determined. In such cases the usual sequence specific primers hybridizing to each strand of a restriction fragment to be amplified can not be defined and therefore one cannot use the methods known in the art for amplification purposes.
[0015]The method of the invention can be used for instance in two different ways, leading to two different types of applications:
[0016](1) Methods for DNA fingerprinting of genomes by randomly selecting subsets of one or more restriction fragments to be amplified by the PCR technique. The invention also covers synthetic oligonucleotides for use in said methods and some applications of said methods can be forensic typing, microbial identification, varietal identification, pedigree analysis and screening of DNA markers linked to genetic traits;
[0017](2) Methods for identifying one or more preselected DNA fragments which can be polymorphic, by PCR amplification. The Invention also covers specific synthetic oligonucleotides for use in said methods and some applications of said methods can be the screening of genetically inherited diseases in humans, monitoring the inheritance of agronomic traits in plant and animal breeding and the detection of infections agents in diseases.

Problems solved by technology

In such cases the usual sequence specific primers hybridizing to each strand of a restriction fragment to be amplified can not be defined and therefore one cannot use the methods known in the art for amplification purposes.

Method used

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  • Selective restriction fragment amplification: fingerprinting
  • Selective restriction fragment amplification: fingerprinting
  • Selective restriction fragment amplification: fingerprinting

Examples

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Effect test

example 1

Selective Restriction Fragment Amplification of Tomato DNA Using PstI

A) Isolation and Modification of the DNA

[0081]Total Tomato DNA (Lycopersicon esculentum c.v. Moneymaker) was isolated from young leaves as described by Bernatzski and Tanksley (Theor. Appl. Genet. 72, 314-321). The typical yield was 50-100 μg DNA per gram of fresh leaf material. The DNA was restricted with PstI (Pharmacia) and double-stranded (ds) PstI-adapters were ligated to the restriction fragments following the procedure described below. These adapters had the following structure (SEQ ID NOS:1-2):

5- CTCCTAGACTGCGTACATGCA -33- CATCTGACGCATGT -5

The 3′TGCA-overhang in these adapters anneals to the staggered ends created by PstI. The PstI recognition sequence CTGCAG is not restored upon ligation of this adapter, because the 5′ C-residue is replaced by A. The ligation reaction was designed in such a way that the end result is almost exclusively DNA fragment-to-adapter molecules. This was achieved by: 1. using non-p...

example 2

Selective Restriction Fragment Amplification of Tomato DNA with Two Restriction Enzymes

[0101]In example 1 the principle of selective restriction fragment amplification (SRDA) is exemplified using Tomato DNA and the restriction enzyme PstI. In this example SRFA using two different restriction enzymes, PstI and MseI, will be illustrated.

Isolation and Modification of the DNA

[0102]Total Tomato DNA was isolated from young leaves as described in example 1. Two pairs of so called isogenic lines were used as source of the DNA, name GemR and GemS, and CGR26 and GCR151 respectively (These lines are described in the following references: Denby and Williams, (1962), Can. J. Plant Sci. 42, 601-685, Smith and Ritchie, (1983), Plant Mol. Biol. Rep. 1, 41-45). The two individuals of each pair of isogenic lines are genetically very similar, but differ in the presence of a trait confering resistance to the fungal pathogen Verticuillium albo-atratum.

[0103]The first step of the modification of the DNA...

example 3

Selective Restriction Fragment Amplification of DNA of Various Lactuca Species with Two Restriction Enzymes

[0131]In example 2 the principle of selective restriction fragment (SRFA) amplification using two restriction enzymes is exemplified for Tomato DNA. In this example we will illustrate that similar results are obtained using DNAs of various Lactuca species using the same two restriction enzymes PstI and MseI.

Isolation and Modification of the DNA

[0132]DNAs were isolated as described in example 1 using young leaf material of various Lactuca species. As indicated below these plants include a commercial lettuce (L. sativa) variety, and several individuals of two wild Lactuca species, L. saligna and L. virosa. The plants were arbitrarily designated the following names:

1. L. saligna, nr. 21, plant 1

2. L. saligna, nr. 21, plant 2

3. L. saligna, nr. 22, plant 1

4. L. saligna, nr. 22, plant 2

5. L. virosa, nr. 01, plant 1

6. L. virosa, nr. 01, plant 2

7. L. virosa, nr. 02,

8. L. virosa, nr. 03...

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Abstract

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.

Description

[0001]This application is a divisional of application Ser. No. 08 / 769,450, filed Dec. 19, 1996, which is a divisional of application Ser. No. 08 / 180,470, filed Jan. 12, 1994, which is a continuation of application Ser. No. 07 / 950,011, filed Sep. 24, 1992.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]This invention relates to applications of DNA fingerprinting and the use of DNA markers in a number of different fields including, but not limited to, plant and animal breeding, variety or cultivar identification, diagnostic medicine, disease diagnosis in animals and plants, identification or genetically inherited disease in humans, family relationship analysis, forensic analysis, and microbial typing.[0004]More specifically, this invention relates to methods for DNA fingerprinting and for detecting specific DNA markers in genomes ranging from microorganisms to higher plants, animals and humans. The invention also relates to synthetic DNA molecules and products based th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07H21/00
CPCC12Q2600/156C12Q1/6876C12Q1/6855C12Q2521/301C12Q2521/501C12Q2525/155C12Q2535/138C12Q2537/159
Inventor ZABEAU, MARCVOS, PIETER
Owner KEYGENE NV
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