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Amplicon melting is a closed-tube method for genotyping that does not require probes , real-time analysis , or allele-specific polymerase chain reaction. However , correct differentiation of homozygous mutant and wild-type samples by melting temperature (T m ) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood , amplicon T m differences of 0.03 to 0.39°C attributable to the extractions were observed. To correct for solution chemistry differences between samples , complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. Highand low-temperature controls bracketing the amplicon melting region decreased the T m SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included , whereas a 3% error rate was observed without temperature correction. In conclusion , internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments. Amplicon melting analysis is a simple closed-tube genotyping method that uses a saturating DNA binding dye instead of fluorescently labeled primers or probes.1 Highresolution melting analysis can detect single base changes and other variations in single or multiplex polymerase chain reaction (PCR).2 Wild-type and homozygous mutant samples typically have sharp, symmetric melting transitions, whereas heterozygous samples have more complex, gradual melting curves. Homozygous sequence changes result in characteristic shifts in melting temperature (T m ). [2][3][4][5] In contrast, heterozygous samples are identified by melting peak shape and width and not by T m . Correct identification of sample genotype by amplicon melting requires standardization of reaction conditions to achieve reproducible, characteristic melting profiles. Reaction conditions can vary between lots of PCR reagents, including different buffers introduced by the DNA isolation method. Ionic strength, in particular, significantly affects T m . -10The current study introduces the use of one or more internal controls for temperature calibration between reactions. Complimentary, unlabeled oligonucleotides that do not interfere with the PCR were designed so that they melt outside the temperature region of PCR product melting. Any buffer differences that affect duplex T m s affects both the amplicon and the internal temperature controls, allowing subsequent temperature correction of melting profiles. As a genotyping target, the 1298AϾC and 677CϾT variants of the methylenetetrahydrofolate reductase (MTHFR) gene were used. A single-color duplex amplicon melting assay (with a...

Elements of the Tx1L family are non-long terminal repeat retrotransposons (NLRs) that are dispersed in the genome of Xenopus laevis. Essentially all genomic copies of Tx1L are found inserted at a specific site within another family of transposable elements (Tx1D). This suggests that Tx1L is a site-specific retrotransposon. Like many (but not all) other NLRs, the Xenopus element encodes an apparent endonuclease that is related in sequence to the apurinic-apyrimidinic endonucleases that participate in DNA repair. This enzyme is thought to introduce the single-strand break in target DNA that initiates transposition by the target-primed reverse transcription (TPRT) mechanism. To explore the issue of target specificity more fully, we expressed the polypeptide encoded by the endonuclease domain of open reading frame 2 from Tx1L (Tx1L EN) and characterized its cleavage capabilities. This endonuclease makes a specific nick in the bottom strand precisely at one end of the presumed Tx1L target duplication. Because this activity leaves a 5-phosphate and 3-hydroxyl at the nick, it has the location and chemistry required to initiate new insertion events by TPRT. Tx1L EN does not make a specific cut at a preferred target site for Tx1D elements, ruling out the alternative possibility that the composite Tx1L-Tx1D element moves as a unit under the control of functions encoded by Tx1L. Further characterization revealed that the endonuclease remains active for many hours at room temperature and that it is capable of enzymatic turnover. Scanning substitution mutagenesis located the recognition site for Tx1L EN within 10 bp surrounding the primary nick site. Implications of these features for natural transposition events are discussed.Transposons are ubiquitous mobile genetic elements found in the genomes of most, if not all, organisms. They can be grouped into two main categories based on sequence organization and mode (or presumed mode) of transposition (3). The first group of transposons consists of the cut-and-paste elements, which move strictly through DNA intermediates. Examples of this type of transposon include the bacterial insertion sequences, the eukaryotic Tc1/Mariner elements, maize Ac/Ds elements, and Drosophila P elements (8,20,24,29,32). The second group, the retrotransposons, transpose through an RNA intermediate.The retrotransposons can be further subdivided into two subgroups that differ in sequence organization and mechanisms of retrotransposition. The retrovirus-like long terminal repeat (LTR) retrotransposons reverse transcribe their RNA genome in the cytoplasm, producing a double-stranded DNA copy with terminal direct repeats. This species is transported to the cell nucleus, where it is integrated into chromosomal DNA courtesy of an element-encoded integrase. Examples of this type of retrotransposon are Ty1 and Ty3 of Saccharomyces cerevisiae, Copia and 412 from Drosophila, and Tf1 from Schizosaccharomyces pombe (3).Although they also rely on reverse transcription, the non-LTR retrotransposons (NLRs) transpose...

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Alport Syndrome is a progressive renal disease with cochlear and ocular involvement. The most common form (~80%) is inherited in an X-linked pattern. X-linked Alport Syndrome (XLAS) is caused by mutations in the type IV collagen alpha chain 5 (COL4A5). We have developed a curated disease-specific database containing reported sequence variants in COL4A5. Currently the database archives a total of 520 sequence variants, verified for their position within the COL4A5 gene and named following standard nomenclature. Sequence variants are reported with accompanying information on protein effect, classification of mutation vs. polymorphism, mutation type based on the first description in the literature, and links to pertinent publications. In addition, features of this database include disease information, relevant links for Alport syndrome literature, reference sequence information, and ability to query by various criteria. On-line submission for novel gene variants or updating information on existing database entries is also possible. This free online scientific resource was developed with the clinical laboratory in mind to serve as a reference and repository for COL4A5 variants.