Amplification of simple sequence repeats
First Claim
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1. A kit for amplification of at least one restriction fragment comprising a simple sequence repeat from a target DNA which comprises at least(a) one double stranded oligonucleotide adaptor having one end matching the ends produced by one targeting restriction enzyme cleaving at or near its recognition sequence which overlaps or flanks the simple sequence repeat;
- and (b) one primer having a sequence comprising;
(i) at the 5′
end of said one primer, a sequence corresponding to the double stranded adaptor or a part thereof; and
(ii) at the 3′
end of said one primer, at least 5 nucleotides corresponding to the sequence of one simple sequence repeat unit or a part thereof.
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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.
54 Citations
6 Claims
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1. A kit for amplification of at least one restriction fragment comprising a simple sequence repeat from a target DNA which comprises at least
(a) one double stranded oligonucleotide adaptor having one end matching the ends produced by one targeting restriction enzyme cleaving at or near its recognition sequence which overlaps or flanks the simple sequence repeat; - and
(b) one primer having a sequence comprising;
(i) at the 5′
end of said one primer, a sequence corresponding to the double stranded adaptor or a part thereof; and
(ii) at the 3′
end of said one primer, at least 5 nucleotides corresponding to the sequence of one simple sequence repeat unit or a part thereof.- View Dependent Claims (3, 4, 5)
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2. A kit for amplification of at least one restriction fragment comprising a simple sequence repeat from a target DNA which comprises at least
(a) two or more restriction enzymes, of which at least one of said two or more restriction enzymes is a targeting enzyme cleaving or at near its recognition sequence which overlaps or flanks the simple sequence repeat and at least one of said two or more restriction enzymes is a frequent cutting enzyme; -
(b) at least two different double stranded oligonucleotide adaptors having one end matching the ends produced by the corresponding restriction enzymes present in said kit;
(c) at least one primer having a sequence comprising;
(i) at the 5′
end of said one primer, a sequence corresponding to the double stranded adaptor corresponding to said targeting enzyme or a part thereof;
and(ii) at the 3′
end of said one primer at least 5 nucleotides corresponding to the sequence of one simple sequence repeat unit or a part thereof; and
(d) at least one primer having a sequence at the 5′
end matching the sequence of the adaptor corresponding to said frequent cutting enzyme and a recognition sequence of said frequent cutting enzyme, or part thereof, and at its 3′
end ranging from 0 to 4 randomly chosen nucleotides.- View Dependent Claims (6)
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Specification
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Current AssigneeKeygene N.V.
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Original AssigneeKeygene N.V.
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InventorsKuiper, Martin T. R., Vos, Pieter, Zabeau, Marc
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Primary Examiner(s)WHISENANT, ETHAN C
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Application NumberUS09/195,991Time in Patent Office879 DaysField of Search435/6, 435/91.2, 435/76, 435/77, 435/78, 536/23.1, 536/24.3US Class Current435/6.12CPC Class CodesC12Q 1/6827 for detection of mutation o...C12Q 1/6855 Ligating adaptorsC12Q 2521/301 EndonucleaseC12Q 2525/151 repeat or repeated sequence...
