Tasting Phenylthiocarbamide (PTC): A New Integrative Genetics Lab with an Old Flavor

First reported in the early 1930s, variation in the ability to taste phenylthiocarbamide (PTC) has since become one of the most widely studied of all human genetic traits. Guo and Reed (2001) provide an excellent review of work on this polymorphism prior to the identification and sequencing of the PTC gene by Kim et al. (2003), and Wooding (2006) provides a stimulating historical review of the role various scientists have played in the study of PTC taste sensitivity and the importance of these studies in relation to natural selection. Identification of the PTC gene and a number of subsequent publications (Wooding et al., 2004; Kim et al., 2005; Wooding et al., 2006) have provided the basis for a new, integrative laboratory investigation of PTC taste sensitivity. This genetics laboratory culminates in the use of the polymerase chain reaction (PCR) and restriction endonuclease digestion to determine the PTC genotype of each student. But “getting there is half the fun” and, in this case, “getting there” requires students to use not only their knowledge of molecular techniques in genetics but also their knowledge of Mendelian genetics, population genetics, probability, and pedigree analysis. The other “half the fun” in this case is that in determining their PTC phenotypes and genotypes, students are learning something about themselves

Human Xq28 Inversion Polymorphism: From Sex Linkage to Genomics - A Genetic Mother Lode

An inversion polymorphism of the filamin and emerin genes at the tip of the long arm of the human X-chromosome serves as the basis of an investigative laboratory in which students learn something new about their own genomes. Long, nearly identical inverted repeats flanking the filamin and emerin genes illustrate how repetitive elements can lead to alterations in genome structure (inversions) through nonallelic homologous recombination. The near identity of the inverted repeats is an example of concerted evolution through gene conversion. While the laboratory in its entirety is designed for college level genetics courses, portions of the laboratory are appropriate for courses at other levels. Because the polymorphism is on the X-chromosome, the laboratory can be used in introductory biology courses to enhance understanding of sex-linkage and to test for Hardy-Weinberg equilibrium in females. More advanced topics, such as chromosome interference, the molecular model for recombination, and inversion heterozygosity suppression of recombination can be explored in upper-level genetics and evolution courses. DNA isolation, restriction digests, ligation, long PCR, and iPCR provide experience with techniques in molecular biology. This investigative laboratory weaves together topics stretching from molecular genetics to cytogenetics and sex-linkage, population genetics and evolutionary genetics

Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design

The soil transmitted helminths are a group of parasitic worms responsible for extensive mor- bidity in many of the world’s most economically depressed locations. With growing empha- sis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing- based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays

A PCR Assay for the Detection of Wuchereria bancrofti in Blood

To identify Wuchereria bancrofti DNA sequences that could be used as the basis for a simple and rapid parasite detection assay, a genomic library of W. bancrofti was constructed and screened for highly repeated DNA. The repeat found with the highest copy number was 195 basepairs (bps) long, 77% AT, and 300 copies per haploid genome. This sequence was designated the Ssp I repeat because it has a unique recognition site for that restriction endonuclease in all or most of the repeat copies. The Ssp I repeat DNA family is dispersed, genus-specific, and exists in all of the different geographic isolates of W. bancrofti tested. Based on DNA sequence analysis of this repeat, we have developed an assay to detect very small quantities of W. bancrofti DNA using the polymerase chain reaction (PCR). With this PCR assay, the Ssp I repeat was detected in as little as 1 pg of W. bancrofti genomic DNA (about 1% of the DNA in one microfilaria) added to 100 pA of human blood. The PCR assay also amplified Ssp I repeat DNA from geographic isolates of W. bancrofti from around the world but not from other species of filariae or from human or mosquito DNA. Microfilaria-positive human blood samples collected in Mauke, Cook Islands were shown to be Ssp I PCR-positive, while microfilaria-negative samples were PCR-negative. The specificity and sensitivity of the Ssp I PCR assay indicates that this approach has significant potential for improved screening of large human populations for active W. bancrofti infection

Comparative assay results for each species of parasite.

<p>Comparative assay results for each species of parasite.</p

Estimated Minimum Quantities of DNA in the Eggs of Each Species of STH.

<p>Estimated Minimum Quantities of DNA in the Eggs of Each Species of STH.</p

Comparative probe testing.

<p>For each novel probe design, FAM-TAMRA and double quenched FAM-ZEN-IOWA BLACK probes were synthesized. Comparative testing revealed that double quenched probes outperformed traditional probes, as evidenced by lower Ct values and greater ΔRn values. The plot above demonstrates these findings with the amplification of three concentrations of <i>N</i>. <i>americanus</i> template DNA using both double quenched (yellow) and traditional (blue) probe designs.</p

Selected primer and probe sequences for each multi-parallel assay.

<p>Selected primer and probe sequences for each multi-parallel assay.</p

Illustrative output from RepeatExplorer analysis of <i>Necator americanus</i>.

<p>During “clustering” each nucleotide within a cluster is assigned a number. That number corresponds to how many individual next-generation sequencing reads that particular nucleotide appeared in. Using this output, a stretch of the most abundant nucleotides (depicted in green within the larger cluster’s sequence) is selected, and the corresponding nucleotides (highlighted in yellow) are selected as the candidate sequence from which the primers and probe are designed.</p

Workflow for repeat analysis.

<p>Output data from a next-generation sequencing run are uploaded to the RepeatExplorer Galaxy-based platform. During the QC and manipulation phase, the <i>FASTQ Groomer</i> tool is used to convert sequence reads into Sanger format. The <i>FASTQ</i>: <i>READ QC</i> tool is then used to verify the quality of the reads before removing unnecessary sequence (i.e. adapter sequences, etc.) from the ends of each read using the <i>FASTQ Trimmer</i> tool. The QC analysis is then repeated, and the <i>FASTQ to FASTA converter</i> tool is used to convert each read into FASTA format. Using these DNA sequence reads as input, sequences undergo clustering, during which an “all-to-all” sequence comparison is performed, and similar sequences are grouped together into clusters. Clusters containing the most highly repetitive sequences are then selected as putative diagnostic targets to be used for primer and probe-based real-time PCR assay design.</p