Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
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Abstract
The present invention relates to a method for identifying a target nucleotide sequence. This method involves forming a ligation product on a target nucleotide sequence in a ligase detection reaction mixture, amplifying the ligation product to form an amplified ligation product in a polymerase chain reaction (PCR) mixture, detecting the amplified ligation product, and identifying the target nucleotide sequence. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence differences.
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Citations
17 Claims
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1-9. -9. (canceled)
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10. A method for identifying copy number in a target nucleotide sequence comprising:
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providing a sample containing one or more target nucleotide sequences potentially comprising one or more copy number differences;
performing a ligase detection reaction to form a ligation product comprising a first target specific portion complementary to the target nucleotide sequence and a second target specific portion complementary to the target nucleotide sequence, wherein the ligation product further comprises a 5′
upstream primer-specific portion and a 3′
downstream primer-specific portion, wherein the first target specific portion and the second target specific portion are located between the 3′
downstream primer-specific portion and the 5′
upstream primer-specific portion;
providing an oligonucleotide primer set comprising (a) an upstream primer containing the same sequence as the 5′
upstream primer-specific portion of the ligation product sequence and (b) a downstream primer complementary to the 3′
downstream primer-specific portion of the ligation product sequence;
performing a polymerase chain reaction mixture to form extension products comprising the ligation product sequence and/or complements thereof; and
,detecting the extension products to identify the copy number of the target nucleotide sequence in the sample. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17)
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Specification