Development of a capillary electrophoresis-based heteroduplex tracking assay to measure in-host genetic diversity of initial and recurrent Plasmodium vivax infections in Cambodia.

A heteroduplex tracking assay used to genotype Plasmodium vivax merozoite surface protein 1 was adapted to a capillary electrophoresis format, obviating the need for radiolabeled probes and allowing its use in settings where malaria is endemic. This new assay achieved good allelic discrimination and detected high multiplicities of infection in 63 P. vivax infections in Cambodia. More than half of the recurrent parasitemias sampled displayed identical or highly related genotypes compared to the initial genotype, suggesting that they represented relapses.

Study of VSX1 mutations in patients with keratoconus in southwest Iran using PCR-single-strand conformation polymorphism/heteroduplex analysis and sequencing method.

OBJECTIVE: Keratoconus (KC) is an eye disorder in which the cornea is swollen, thinned and deformed. Despite extensive studies, the pathophysiological processes and genetic etiology of KC are unknown. The disease incidence is approximately 1 in 2,000, and it is the most common cause of corneal transplantation in the USA. Many genes are involved in the disease, but evidence suggests a major role for VSX1 in the etiology of KC. This study aimed to determine the frequency of mutations in exons 2, 3 and 4 of the VSX1 gene in Chaharmahal va Bakhtiari province in the southwest of Iran. STUDY DESIGN: In this experimental study, mutations in 3 exons, namely exons 2, 3 and 4, of VSX1 were investigated in 50 patients with KC and 50 healthy control subjects. DNA was extracted using a standard phenol-chloroform method. PCR-single-strand conformational polymorphism/heteroduplex analysis was performed, followed by DNA sequencing to confirm the identified motility shifts. RESULTS: H244R mutations were found in 1 patient and also in 1 healthy control subject. Furthermore, 12 polymorphisms were identified in patients with KC and 7 in healthy control subjects [rs6138482 and c.546A>G (rs12480307)]. CONCLUSION: Our investigation showed that KC-related VSX1 mutations were found in a very small proportion of the studied patients from Iran. Further investigations on other genes are needed to clarify their roles in KC pathogenesis.

Combining results of forensic STR kits: HDplex validation including allelic association and linkage testing with NGM and Identifiler loci.

The autosomal short tandem repeat (STR) kits that are currently used in forensic science have a high discrimination power. However, this discrimination power is sometimes not sufficient for complex kinship analyses or decreases when alleles are missing due to degradation of the DNA. The Investigator HDplex kit contains nine STRs that are additional to the commonly used forensic markers, and we validated this kit to assist human identification. With the increasing number of markers it becomes inevitable that forensic and kinship analyses include two or more STRs present on the same chromosome. To examine whether such markers can be regarded as independent, we evaluated the 30 STRs present in NGM, Identifiler and HDplex. Among these 30 markers, 17 syntenic STR pairs can be formed. Allelic association between these pairs was examined using 335 Dutch reference samples and no linkage disequilibrium was detected, which makes it possible to use the product rule for profile probability calculations in unrelated individuals. Linkage between syntenic STRs was studied by determining the recombination fraction between them in five three-generation CEPH families. The recombination fractions were compared to the physical and genetic distances between the markers. For most types of pedigrees, the kinship analyses can be performed using the product rule, and for those cases that require an alternative calculation method (Gill et al., Forensic Sci Int Genet 6:477-486, 2011), the recombination fractions as determined in this study can be used. Finally, we calculated the (combined) match probabilities, for the supplementary genotyping results of HDplex, NGM and Identifiler.

Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy.

BACKGROUND: Nephronophthisis associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity, a strategy of DNA pooling with consecutive massively parallel resequencing (MPR) was devised. METHODS: In 120 patients with severe NPHP-AC phenotypes, five pools of genomic DNA with 24 patients each were prepared which were used as templates in order to PCR amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on an Illumina Genome-Analyser and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease based heteroduplex screening and confirmed by Sanger sequencing. RESULTS: For proof of principle, DNA from patients with known mutations was used and detection of 22 out of 24 different alleles (92% sensitivity) was demonstrated. MPR led to the molecular diagnosis in 30/120 patients (25%) and 54 pathogenic mutations (27 novel) were identified in seven different NPHP-AC genes. Additionally, in 24 patients only single heterozygous variants of unknown significance were found. CONCLUSIONS: The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single gene disorders. The lack of mutations in 75% of patients in this cohort indicates further extensive heterogeneity in NPHP-AC.

Blinded study determination of high sensitivity and specificity microchip electrophoresis-SSCP/HA to detect mutations in the p53 gene.

Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA-based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor-intensive, time-consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here, we demonstrate the first blinded study using microchip electrophoresis (ME)-SSCP/HA. We demonstrate the ability of ME-SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5-9 in a blinded study in an analysis time of <10 min.

An assay of gene copy number and its application based on heteroduplex products.

The aim of this study was to set up a simple and efficient method for detecting gene copy number, based on heteroduplex products from single-tube PCR/DHPLC. Single-nucleotide polymorphisms (SNPs) on the α-globin gene and chromosome 21 were used as examples. And the formula for quantitative calculation of gene copy number was deduced-based on the peak heights of homoduplexes and heteroduplexes on the DHPLC pattern. 27 samples (14 normal DNA and 13 cases of trisomy-21) were assessed with this method, and 160 samples (48 normal DNA and 112 α-thalassemia samples) were assessed with this method combined with a duplex PCR/DHPLC. Results for 184 of 187 cases were concordant with the known genotypes; three cases of trisomy-21 could not be detected because the target SNPs were homozygous. In conclusion, quantitative assessment of heteroduplex products from single-tube PCR/DHPLC is simple and rapid, and can be used to detect α-thalassemia gene deletions (α(-3.7), α(-4.2)) and trisomy-21.

Investigation of matched and mismatched duplex DNA by electrospray ionization-mass spectrometry.

In this research, the gas-phase stabilities of matched and mismatched duplex DNA were investigated by electrospray ionization-mass spectrometry (ESI-MS). The wild-type p53 duplex DNA [ds1, perfectly-matched (PM) DNA] was successfully distinguished from its three mutated DNAs [double-base mismatched DNA (DM)]. Moreover, the three DM DNAs were also well discriminated from each other using ESI-MS. Results show that the gas-phase thermodynamic stability of the DM DNAs decreased as the two mismatch spots moved closer. This implies that the dissociation of DM duplexes into two single strands prefers the mode "from middle to terminals".

Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing.

Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.

Hepatitis C virus (HCV) quasispecies complexity and selection in HCV/HIV-coinfected subjects treated with interferon-based regimens.

BACKGROUND: Coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) has emerged as a major cause of morbidity and mortality due to liver disease. Interferon-based therapy response rates have been disappointingly low. Baseline HCV complexity and the relationship between complexity and viral kinetic parameters has not been well described in HCV/HIV-coinfected subjects. METHODS: A subset of patients enrolled in the AIDS Clinical Trials Group 5071 trial underwent sampling to evaluate viral kinetics and changes in HCV complexity. Early kinetic parameters, baseline complexity, and treatment outcomes--including rapid viral response (RVR), early viral response (EVR), and sustained viral response (SVR)--were evaluated. HCV-monoinfected subjects were matched to HCV/HIV-coinfected subjects. RESULTS: Baseline complexity was determined in 108 HCV/HIV-coinfected subjects and in 13 HCV-monoinfected control subjects. Quasispecies complexity was a mean of 2.24 bands for HCV/HIV-coinfected subjects and a mean of 1.90 bands for HCV-monoinfected subjects (P = .14). Lower baseline complexity was associated with EVR (P = .04) and approached statistical significance for SVR. In patients who underwent viral kinetic modeling, a decrease in complexity was associated with RVR (P = .03) and was independent of the correlation between first-phase viral decline efficiency and RVR. CONCLUSION: Baseline HCV complexity is an independent predictor of EVR in HCV/HIV-coinfected subjects. A decrease in complexity occurs by 4 weeks after the initiation of interferon-based therapy and is associated with RVR. These findings may enhance the predictive modeling of treatment outcome in HCV/HIV-coinfected patients.

PRIMA: a rapid and cost-effective genotyping method to detect single-nucleotide differences using probe-induced heteroduplexes.

Targeted mutagenesis by programmable site-specific nucleases like CRISPR typically produce 1-base pair (bp) insertion or deletion (indel) mutations. Although several methods have been developed to detect such 1-bp indels, each method has pros and cons in terms of cost and/or resolution. Heteroduplex mobility assay (HMA) is a traditional technique detecting small base pair differences but it has a limited resolution of mutation size and the band patterns are often complex. Here, we developed a new method called PRIMA (Probe-Induced HMA) using a short single-stranded DNA molecule as a probe in HMA. By utilizing a 40-mer probe containing a 5-nucleotide deletion, we assessed the mobility of a heteroduplex with a target DNA fragment from a plant, bacterium, and human. This method allowed us to detect a 1-bp indel mutation consistently. We also showed that SNPs can be detected using PRIMA. PRIMA provides a rapid and cost-effective solution for the genotyping.

Consecutive terminal GU pairs stabilize RNA helices.

Consecutive GU pairs at the ends of RNA helices provide significant thermodynamic stability between -1.0 and -3.8 kcal/mol at 37 °C, which is equivalent to approximately 2 orders of magnitude in the value of a binding constant. The thermodynamic stabilities of GU pairs depend on the sequence, stacking orientation, and position in the helix. In contrast to GU pairs in the middle of a helix that may be destabilizing, all consecutive terminal GU pairs contribute favorable thermodynamic stability. This work presents measured thermodynamic stabilities for 30 duplexes containing two, three, or four consecutive GU pairs at the ends of RNA helices and a model to predict the thermodynamic stabilities of terminal GU pairs. Imino proton NMR spectra show that the terminal GU nucleotides form hydrogen-bonded pairs. Different orientations of terminal GU pairs can have different conformations with equivalent thermodynamic stabilities. These new data and prediction model will help improve RNA secondary structure prediction, identification of miRNA target sequences with GU pairs, and efforts to understand the fundamental physical forces directing RNA structure and energetics.

Identification and recovery of minor HIV-1 variants using the heteroduplex tracking assay and biotinylated probes.

We describe a method to identify and recover minor human immunodeficiency virus type 1 (HIV-1) sequence variants from a complex population. The original heteroduplex tracking assay (HTA) was modified by incorporating a biotin tag into the probe to allow for direct sequence determination of the query strand. We used this approach to recover sequences from minor HIV-1 variants in the V3 region of the env gene, and to identify minor drug-resistant variants in pro. The biotin-HTA targeting of the V3 region of env allowed us to detect minor V3 variants, of which 45% were classified as CXCR4-using viruses. In addition, the biotin-protease HTA was able to detect mixtures of wild-type sequence and drug-resistance mutations in four subjects that were not detected by bulk sequence analysis. The biotin-HTA is a robust assay that first separates genetic variants then allows direct sequence analysis of major and minor variants.

Heteroduplex polymerase chain reaction is essential for canine receptor rearrangement analysis.

Polymerase chain reaction (PCR) for antigen receptor rearrangement is a sensitive technique for detecting lymphocyte-proliferative disorders, but it tends to produce false-positive results, a phenomenon termed pseudoclonality. Heteroduplex analysis, which is useful to distinguish clonal reactions from pseudoclonal ones in dogs, can be applied to avoid misdiagnosis and determine the reliability of results. In the current study, PCR for antigen receptor rearrangement was used to identify clonal proliferation of lymphocytes in duodenal and lymphoid tissue from dogs presenting with chronic vomiting and enlarged peripheral lymph nodes typical of multicentric lymphoma, and the test results were verified with heteroduplex analysis. In the case of almost all of the duodenal samples, even without a histologic diagnosis of lymphoma, a distinct band similar to that observed in the case of lymphoma was obtained for both B- and T-cell clonality. All of the bands obtained from the nonneoplastic duodenum disappeared following heteroduplex analysis of the PCR product, whereas the distinct bands from the lymphoma remained. In the lymph node samples, the pseudoclonal bands that disappeared in the heteroduplex analysis were detected mainly in B cells. In conclusion, heteroduplex analysis with PCR for antigen receptor rearrangement is a suitable tool for diagnosing canine lymphoma and decreasing the possibility of misdiagnosis of pseudoclonality.

Investigation of putative multisubtype hepatitis C virus infections in vivo by heteroduplex mobility analysis of core/envelope subgenomes.

The frequency that multiple different subtypes of hepatitis C virus (HCV) simultaneously infect a given individual is controversial. To address this question, heteroduplex mobility analysis (HMA) of portions of the HCV core and envelope 1 region was optimized for sensitive and specific detection of mixtures of HCV genomes of different genotype or subtype. Using the standard HCV genotyping approach of 5'-untranslated region (UTR) analysis, 28 of 374 (7.5%) chronic hepatitis C research subjects were classified as having either multiple-subtype HCV infections (n = 21) or switching HCV subtypes over time (n = 7), the latter pattern implying viral superinfection. Upon retesting of specimens by HMA, 25 of 28 multiple-subtype results could not be reproduced. All three patients with positive results were injection drug users with potential multiple HCV exposures. To address the hypothesis of tissue sequestration of multiple-subtype HCV infections, liver (n = 22), peripheral blood mononuclear cell (n = 13), perihepatic lymph node (n = 16), and serum (n = 19) specimens from 23 subjects with end-stage hepatitis C were collected and analyzed by the HMA technique. Whereas 5'-UTR results implicated mixed-subtype HCV infections in 2 subjects, HMA testing revealed no evidence of a second HCV subtype in any tissue compartment (0 of 70 compartments [0%]) or within any given subject (0 of 23 subjects [0%]). In summary, a large proportion of mixed-genotype and switching-genotype patterns generated by 5'-UTR analysis were not reproducible using the HMA approach, emphasizing the need for additional study.

Determination of DNA mutation load in human tissues using denaturing HPLC-based heteroduplex analysis.

Since its introduction more than a decade ago, denaturing HPLC has been widely used to screen nuclear and mitochondrial DNA for mutations. We recently developed a quantitative method based on denaturing HPLC to measure DNA mutation load, using tRNA Leu(UUR) region of the mitochondrial DNA as an example. The mutation load is determined based on the quadratic function that governs DNA reannealing and the formation of heteroduplex and homoduplex DNAs. We have used this assay to measure heteroplasmy level for several mitochondrial mutations in DNAs obtained from human tissue samples. This quantitative DHPLC assay is well suited for simultaneous detection and quantification of DNA mutations.

A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons.

We describe a rapid, high-throughput method to scan for new RNA editing sites. This method is adapted from high-resolution melting (HRM) analysis of amplicons, a technique used in clinical research to detect mutations in genomes. The assay was validated by the discovery of six new editing sites in different chloroplast transcripts of Arabidopsis thaliana. A screen of a collection of mutants uncovered a mutant defective for editing of one of the newly discovered sites. We successfully adapted the technique to quantify editing of partially edited sites in different individuals or different tissues. This new method will be easily applicable to RNA from any organism and should greatly accelerate the study of the role of RNA editing in physiological processes as diverse as plant development or human health.

An improved heteroduplex analysis for rapid genotyping of SNPs and single base pair indels.

SNPs and single base pair (SBP) insertion/deletions (indels) are not only the most abundant genetic markers for genetic mapping and breeding selection, but also always occur in the mutants generated from chemical mutagenesis or CRISPR/Cas9-mediated genome editing. Most of the current SNP and SBP indel genotyping methods are time-consuming and/or require special equipment or reagents. Here, we describe an improved heteroduplex analysis method, named iHDA, that can readily discriminate SNP and SBP indel alleles with specially designed DNA probes that harbor a couple of nucleotides adjacent to the SNP site. By hybridizing with the same probe, SNP and SBP indel alleles form different heteroduplexes, differing in bulge size, which show different mobility on a polyacrylamide gel. Therefore, iHDA is an easy, fast and inexpensive method for SNP and SBP indel genotyping.

PCR GeneScan and Heteroduplex Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes for Clonality Diagnostics in Suspect Lymphoproliferations.

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) or consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium. The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan and heteroduplex analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.

Comparison of the mismatch-specific endonuclease method and denaturing high-performance liquid chromatography for the identification of HBB gene mutations.

BACKGROUND: Beta-thalassemia is a common autosomal recessive hereditary disease in the Meditertanean, Asia and African areas. Over 600 mutations have been described in the beta-globin (HBB), of which more than 200 are associated with a beta-thalassemia phenotype. RESULTS: We used two highly-specific mutation screening methods, mismatch-specific endonuclease and denaturing high-performance liquid chromatography, to identify mutations in the HBB gene. The sensitivity and specificity of these two methods were compared. We successfully distinguished mutations in the HBB gene by the mismatch-specific endonuclease method without need for further assay. This technique had 100% sensitivity and specificity for the study sample. CONCLUSION: Compared to the DHPLC approach, the mismatch-specific endonuclease method allows mutational screening of a large number of samples because of its speed, sensitivity and adaptability to semi-automated systems. These findings demonstrate the feasibility of using the mismatch-specific endonuclease method as a tool for mutation screening.

Heteroduplex analysis using flow cytometric microbead assays to detect deletions, insertions, and single-strand lesions.

We explore the possibilities offered by flow cytometric microbead analysis to develop high throughput methods for the detection of deletions/insertions and single-strand DNA lesions. The products of PCR reactions derived from reference and test samples are denatured and reannealed, then exposed to enzymatic or chemical treatments distinguishing homoduplices from heteroduplices. The biotin- and dye labeled reaction products are immobilized on microbeads and the homo- and heteroduplices are assessed in separate fluorescence channels, by flow cytometry. Using a model system based on the mixed lineage leukemia gene breakpoint cluster region, we demonstrate that deletions and insertions in genomic DNA can be detected, using S1 nuclease and chemical cleavage to distinguish hetero- from homoduplices, or a restriction enzyme cleaving only the homoduplices. Single-strand discontinuities can also be detected, by combining nick-translation, using labeled nucleotide, and flow cytometric microbead analysis. The methodical approaches demonstrated are applicable in a versatile manner in basic cell and molecular biological research and also promise direct application for high throughput screening of genetic diseases and lesions, including insertions or deletions of short sequence elements and single-strand lesions formed at hypersensitive sites in response to apoptotic stimuli.