Method for detecting mutations in nucleic acids

Details Method for detecting mutations in nucleic acids Method for detecting mutations in nucleic acids

1999-12-30

2001-11-06

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C436S094000, C436S501000, C536S026110, C536S027400, C536S028500, C536S029200

Reexamination Certificate

active

06312905

ABSTRACT:

BACKGROUND OF THE INVENTION
Mutation scanning methods are important for elucidating the genetic basis of human disease. Single-stranded conformation polymorphism (SSCP)(7) (a bibliography is provided at the end of the written description) is the most widely used mutation scanning method, but its sensitivity varies. Two hybrids between SSCP and Sanger dideoxy sequencing have been developed. These hybrid methods can detect the presence of virtually all mutations. The first hybrid method is dideoxy fingerprinting (ddF). A Sanger reaction is performed with one dideoxy terminator and with one primer to produce a nested set of 5′ co-terminal DNA segments, then the segments are denatured and electrophoresed through a non-denaturing gel (10). Mutations can be detected by an alteration in the mobility of at least one of the multiple termination segments that contain the mutation (informative SSCP component). In addition, 6 out of 12 types of possible single-base substitutions result in a gain and/or loss of a dideoxy termination segment at the mutation site (informative dideoxy component). In manual gels, ddF can detect virtually all mutations in a 300-bp region of DNA (1,5,10). The second hybrid method, bidirectional ddF (Bi-ddF) is a modification of ddF in which a cycle sequencing is performed with opposite primers to scan simultaneously for mutations in both directions. Bi-ddF has two important advantages over ddF: (i) the dideoxy component can detect 10 out of 12 types of possible single-base substitutions, and (ii) the SSCP component is on the average more informative because alterations of mobility can be detected in either the downstream or upstream direction. As a result, Bi-ddF can screen 600-bp segments with virtually 100% sensitivity (2,4). However, when these methods are adapted for high G+C regions, smearing of bands sometimes lowers the resolution.
SUMMARY OF THE INVENTION
In accordance with the present invention, a denaturation fingerprinting method (“dnf”) comprises subjecting a nucleic acid segment to bidirectional cycle sequencing using oppositely oriented primers that bound the segment of interest, wherein two different dideoxynucleotides (ddNTPs) are employed in the sequencing reaction and separating the fragments by denaturing electrophoresis to produce a fingerprint pattern.
In one embodiment, designated dnF
2R
, fingerprints are generated by performing denaturing gel electrophoresis on bidirectional cycle-sequencing reactions with each of two ddNTPs, e.g., ddATP and ddCTP. When the fingerprints are combined, all sequence changes are expected to result in one extra and one absent segment.
An alternative embodiment, designated dnF
1R
, involves performing a single reaction with one ddNTP and a second chemically modified terminator (e.g., ROX-conjugated ddCTP), which retards the mobility of the same termination products, thus permitting resolution of fragments in a single electrophoresis lane.
Denaturation fingerprinting involves technology that is essentially identical to sequencing. Thus, the expertise in sequencing that many laboratories have can be applied directly to these methods. DnF is particularly advantageous for regions of high G+C content, such as promoter regions, because segments may smear when bi-ddF is performed in the absence of urea. However, substantial evidence indicates that increasing the urea concentration to 0.5 M for 60% G+C region and 1.5 M for 70% G+C regions resolves the problem without reducing the sensitivity at those G+C contents. While bi-ddF and dnF may involve the use of urea for high G+C segments bi-ddF utilizes the SSCP effect as the primary mechanism of detecting mutation, while dnF uses the dideoxy component as the primary mechanism of detecting mutations and the SSCP effect is the secondary mode of detecting mutations.


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