Dade Behring Marburg Gmbh

Dade Behring Inc.

Aventis Behring Gmbh

Freshgene, Inc.

Alla Lishanski

Nurith Kurn

Edwin F Ullman

Marc Taylor

Yen Ping Liu

Qinghong Yang

Wendy Yang

The present invention provides methods for detecting the presence or absence of a difference between two related nucleic acid sequences. In the methods, a target nucleic acid and a reference nucleic acid are contacted under conditions in which they are capable of forming a four-way nucleic acid complex with a branch structure that is capable of migration. Under the contact conditions, if the reference nucleic acid and target nucleic acid are identical, branch migration is capable of going to completion resulting in complete strand exchange. If the reference nucleic acid and target nucleic acid are different, branch migration does not go to completion, resulting in a stable four-way complex. Detection of the stable four-way complex identifies the presence of a difference between the nucleic acids. A stable four-way complex can be detected with molecules that specifically bind such complexes, by gel electrophoresis or by specific isolation of the stable four-way complex.

Methods for detection of differences in nucleic acids

A method is disclosed for detecting the presence of a difference between two related nucleic acid sequences. In the method a complex is formed comprising both strands of each sequence. Each member of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of a difference between the two related sequences. The complex generally comprises a Holliday junction. In one aspect a medium suspected of containing said two related nucleic acid sequences is treated to provide partial duplexes having non-complementary tailed portions at one end. The double stranded portions of the partial duplexes are identical except for said difference. One of the strands of one of the partial duplexes is complementary to one of the strands of the other of the partial duplexes and the other of the strands of one of the partial duplexes is complementary to the other of the strands of the other of the partial duplexes. The medium is subjected to conditions that permit the binding of the tailed portions of the partial duplexes to each other. If there is a difference in the related nucleic acid sequences, a stable complex is formed comprising a Holliday junction. If no difference exists, the complex dissociates into duplexes. A determination is made whether the stable complex is formed, the presence thereof indicating the presence of the related nucleic acid sequences. The method has application in detecting the presence of a mutation in a target sequence or in detecting the target sequence itself.

Detection of differences in nucleic acids

The present invention relates to a method for selectively extending an oligonucleotide primer along a specific target polynucleotide sequence in a mixture of polynucleotides. Selective extension is achieved by controlling the concentration of the oligonucleotide primer by forming the oligonucleotide in situ by degrading the 3&prime;-end of a modified oligonucleotide. The method comprises providing in combination the mixture of polynucleotides and the modified oligonucleotide having a 3&prime;-end that is not extendable along any polynucleotide and extending the oligonucleotide primer selectively along the specific target polynucleotide sequence by controlling the degradation of the 3&prime;-end of the modified oligonucleotide. In this way extension of the oligonucleotide primer along polynucleotides other than the specific target polynucleotide sequence is substantially reduced or avoided. In another aspect the invention is an improvement in a method for amplifying a target polynucleotide sequence. The method comprises combining the target polynucleotide sequence with reagents for amplifying the target polynucleotide sequence and subjecting the combination to conditions wherein the target polynucleotide sequence is amplified. The reagents comprise an oligonucleotide primer and a polymerase. The improvement comprises deriving the oligonucleotide primer from a modified oligonucleotide having a portion that hybridizes to the target polynucleotide sequence except for the 3&prime;-end thereof, which has at least one nucleotide analog that is incapable of hybridizing to a polynucleotide. Kits for carrying out the above methods are also disclosed.

Method for polynucleotide amplification

A method is disclosed for detecting the presence of a difference between two related nucleic acid sequences. In the method a complex is formed comprising both strands of each sequence. Each member of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of a difference between the two related sequences. The complex generally comprises a Holliday junction. In one aspect a medium suspected of containing said two related nucleic acid sequences is treated to provide partial duplexes having non-complementary tailed portions at one end. The double stranded portions of the partial duplexes are identical except for said difference. One of the strands of one of the partial duplexes is complementary to one of the strands of the other of the partial duplexes and the other of the strands of one of the partial duplexes is complementary to the other of the strands of the other of the partial duplexes. The medium is subjected to conditions that permit the binding of the tailed portions of the partial duplexes to each other. If there is a difference in the related nucleic acid sequences, a stable complex is formed comprising a Holliday junction. If no difference exists, the complex dissociates into duplexes. A determination is made whether the stable complex is formed, the presence thereof indicating the presence of the related nucleic acid sequences. The method has application in detecting the presence of a mutation in a target sequence or in detecting the target sequence itself. Also provided is an alternative primer scheme which allows for the reduction of background signal due to mis-priming during amplification of the nucleic acid sequences in the detection method described herein.

Detection of differences in nucleic acids by inhibition of spontaneous DNA branch migration

The present invention relates to a method for selectively extending an oligonucleotide primer along a specific target polynucleotide sequence in a mixture of polynucleotides. A combination is provided comprising the mixture, an oligonucleotide primer having a modification, and a binding substance for the modification wherein the binding substance binds to the oligonucleotide and prevents the extension of the oligonucleotide along the target polynucleotide sequence. The temperature of the combination is adjusted to a level sufficient to irreversibly denature the binding substance and permit the extension of the oligonucleotide primer along the specific target polynucleotide sequence. The invention has particular application in the amplification of nucleic acids. Also disclosed are kits for carrying out a method in accordance with the present invention.

Growing community of inventors

San Jose, CA, United States of America

Alla Lishanski