Amplification of simple sequence repeats
First Claim
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1. Process for the selective amplification of restriction fragments comprising simple sequence repeats, comprising the following:
- (a) digesting a starting target DNA with two or more different restriction enzymes, at least one of these enzymes cleaving at or near its recognition nucleotide sequence overlapping or flanking with the simple sequence repeat (referred to as first restriction enzyme) and at least one of these enzymes cleaving the restriction fragments into amplificable restriction fragments (referred to as second restriction enzyme) to obtain restriction fragments,(b) ligating an appropriate double stranded oligonucleotide adaptor to each of the ends of the restriction fragments produced by said restriction enzymes,(c) amplifying the restriction fragments of step b) using two or more different amplification primers with the following general structure;
one primer having a sequence at its 5'"'"' end matching the common sequence of the restriction fragments produced with the first restriction enzyme, and a sequence at its 3'"'"' end of at least 5 nucleotides matching the sequence of the simple sequence repeat (referred to as primer one);
one primer having a sequence at its 5'"'"' end matching the common sequence of the restriction fragments produced with the second restriction enzyme (referred to as primer two),(d) recovering the amplified fragments.
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Abstract
The invention relates to a process for the selective amplification of restriction fragements comprising simple sequence repeats.
The process of the invention can be used in a range of fields including but not limited to plant and animal breeding, genetic identity testing in humans, plants and animals, disease identification and screening, forensic analysis and gene tagging and isolation.
42 Citations
15 Claims
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1. Process for the selective amplification of restriction fragments comprising simple sequence repeats, comprising the following:
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(a) digesting a starting target DNA with two or more different restriction enzymes, at least one of these enzymes cleaving at or near its recognition nucleotide sequence overlapping or flanking with the simple sequence repeat (referred to as first restriction enzyme) and at least one of these enzymes cleaving the restriction fragments into amplificable restriction fragments (referred to as second restriction enzyme) to obtain restriction fragments, (b) ligating an appropriate double stranded oligonucleotide adaptor to each of the ends of the restriction fragments produced by said restriction enzymes, (c) amplifying the restriction fragments of step b) using two or more different amplification primers with the following general structure; one primer having a sequence at its 5'"'"' end matching the common sequence of the restriction fragments produced with the first restriction enzyme, and a sequence at its 3'"'"' end of at least 5 nucleotides matching the sequence of the simple sequence repeat (referred to as primer one); one primer having a sequence at its 5'"'"' end matching the common sequence of the restriction fragments produced with the second restriction enzyme (referred to as primer two), (d) recovering the amplified fragments. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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Specification