INVERSE MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (iMLPA), AN IN VITRO METHOD OF GENOTYPING MULTIPLE INVERSIONS

An in vitro method for detecting the orientation of a genomic sequence within a larger sequence, wherein said genomic sequence is connected to the larger sequence at its 5′ and 3′ ends by a 5′ junction region and by a 3′ junction region in a sample comprising nucleic acids, said method comprising the following steps:

• (i) digesting nucleic acids with at least a restriction enzyme, said restriction enzyme having at least a target site in the genomic sequence flanked by a junction region and at least another target site outside the genomic sequence flanked by a junction region,
• (ii) circularizing the digested nucleic acid fragments obtained in step (i) by self-ligation with a ligase enzyme, thereby generating a circular nucleic acid comprising a junction region and a reconstituted target site for the restriction enzyme used in step (i), said reconstituted target site flanked on one side by the region originally located 3′ with respect to the junction region and on the other side by the region originally located 5′ with respect to the junction region,
• (iii) incubating the circularized nucleic acids obtained in step (ii) with at least a probe pair, each probe pair selected from the group consisting of:
• I. a probe pair comprising:
• a) a first oligonucleotide Having a 5′ region and a 3′ region, wherein the 3′ region of said first oligonucleotide is complementary to a region of the genomic sequence flanked by a junction region and wherein the 3′ end of said first oligonucleotide is phosphorylated and
• b) a second oligonucleotide having a 5′ region and a 3′ region, wherein the 5′ region of said second oligonucleotide is complementary to a region of the larger sequence originally located outside the genomic sequence flanked by a junction region
• and wherein the nucleotide position within the circularized genomic sequence to which the 3′ end of the first oligonucleotide hybridizes and the nucleotide position within the genomic sequence to which the 5′ end of the second oligonucleotide hybridizes are adjacent positions, and
• wherein the region of the circularized genomic sequence to which the first and second oligonucleotide hybridize comprises the target site generated after the ligation step (ii), and
• II. a probe pair comprising:
• a) a first oligonucleotide having a 5′ region and a 3′ region, wherein the 3′ region of said first oligonucleotide is complementary to a region of the genomic sequence originally located outside the genomic sequence flanked by a junction region and wherein the 3′ end of said first oligonucleotide is phosphorylated and
• b) a second oligonucleotide having a 5′ region and a 3′ region, wherein the 5′ region of said second oligonucleotide is complementary to a region of the genomic sequence flanked by a junction region
• and wherein the nucleotide position within the circularized genomic sequence to which the 3′ end of the first oligonucleotide hybridizes and the nucleotide position within the genomic sequence to which the 5′ end of the second oligonucleotide hybridizes are adjacent positions, and
• wherein the region of the circularized genomic sequence to which the first and second oligonuclectide hybridize comprises the target site generated after the ligation step (ii),
• (iv) ligating the 3′ end of the first oligonucleotide with the 5′ end of the second oligonuclectide of each probe pair to form an assembled probe,
• (v) amplifying the assembled probe obtained in step (iv) by using a pair of primers, wherein the forward primer hybridizes to the 5′ region of the first oligonucleotide of the probe pair and the reverse primer hybridizes to the 3′ region of the second oligonucleotide of the probe pair, and
• (vi) detecting the product of step (v).