Fluorescent signatures for variable DNA sequences

Abstract

Life abounds with genetic variations writ in sequences that are often only a few hundred nucleotides long. Rapid detection of these variations for identification of genetic diseases, pathogens and organisms has become the mainstay of molecular science and medicine. This report describes a new, highly informative closed-tube polymerase chain reaction (PCR) strategy for analysis of both known and unknown sequence variations. It combines efficient quantitative amplification of single-stranded DNA targets through LATE-PCR with sets of Lights-On/Lights-Off probes that hybridize to their target sequences over a broad temperature range. Contiguous pairs of Lights-On/Lights-Off probes of the same fluorescent color are used to scan hundreds of nucleotides for the presence of mutations. Sets of probes in different colors can be combined in the same tube to analyze even longer single-stranded targets. Each set of hybridized Lights-On/Lights-Off probes generates a composite fluorescent contour, which is mathematically converted to a sequence-specific fluorescent signature. The versatility and broad utility of this new technology is illustrated in this report by characterization of variant sequences in three different DNA targets: the rpoB gene of Mycobacterium tuberculosis, a sequence in the mitochondrial cytochrome C oxidase subunit 1 gene of nematodes and the V3 hypervariable region of the bacterial 16 s ribosomal RNA gene. We anticipate widespread use of these technologies for diagnostics, species identification and basic research.

AZT Treatment Increases mtDNA Mutations in HepG2 and CCD-1112Sk Cells

“…Instances of more than one mutation in a sequence are indicated by a hyphen between mutations. [29,37,38] and confirmed here in multiple instances by sequencing, were not likely to be the result of amplification errors and were not the result of cloning errors [28]. While the total number of mutations after thirty days of treatment was relatively large, the number of mutations in individual experiments was small (Supplemental Table 2).…”

“…The drawback of this method is that hundreds of individual mitochondrial genomes have to be sequenced to properly measure mutational load. The present paper describes the use of LATE-PCR together with a novel probe design called Lights-On/Lights-Off probes [29] to simultaneously screen multiple loci in individual mitochondrial genomes for sequence changes. These probes generate fluorescent signatures that are unique to each amplified sequence.…”

Closed-Tube Barcoding

“…In the case of the rpoB gene of Mycobacterium tuberculosis, the Limiting Primer, the Excess Primer, and the target sequence are as described in Rice et al 2012. In the case of the CO1 gene of Ixodes scapularis there were three Limiting Primers (5=-ACGACATAGTTATACCTGTAATAATTGGGGGTTTTGG-3=, 5=-CACGACATAGTTATACCAATTATAATCGGAGGATTTGG-3=, 5=-CGACATAGTTATACCTGTTATGATTGGTGGGTTTGG-3=) and two Excess Primers (5=-CTGTAATTAAAACTGATCATAC AAATAAAGGTATTCG-3=, 5=-GCTATGACTGATCATACAAA TAAAGGTATTCG-3=) that were designed by comparison of the CO1 sequences from the 20 species of ticks listed in the supplementary data 2 .…”

“…Fluorescent signatures are easier to "read" than fluorescent contours, as their high and low values are more dramatic and are independent of signal strengths (see Fig. 1; Rice et al 2012).…”

“…In the realm of in vitro diagnostics, it is important not to confuse these variants with the many mutations that cause drug resistance. To accomplish this objective, we coat the target strand with three pairs of Lights-On/Lights-Off probes (for details see Rice et al 2012). All of these Lights-On probes are labelled with Quasar 670.…”