Purified Polymerase Chain Reaction Products Research Articles

Occurrence of Banana bunchy top virus in banana and plantain (Musa sp.) in Benin

Banana bunchy top virus (BBTV; genus Babuvirus, family Nanoviridae), is one of the most serious pathogens of banana (AAA genome) and plantain (AAB genome) (Musa spp). BBTV is well established in central and southern sub-Saharan Africa (Kumar et al., 20011). It is transmitted by the banana aphid, Pentalonia nigronervosa (Homoptera: Aphididae), in a persistent manner. In July 2011, banana and plantain that displayed stunting and leaf symptoms typical of banana bunchy top disease were observed to be widespread in Dangbo Commune, Oueme Department, Benin. To identify the cause of the disease, a roving survey was conducted in December 2011 in nine locations in Avrankou, Dangbo, Akpro-Misserete and Porto-Novo Communes, in Oueme (Fig. 1). In each location, incidence of symptom-bearing plants was estimated from counts of 15 mats, and samples were collected for BBTV assessment. About 60% of the 94 banana mats assessed had plants exhibiting typical symptoms of BBTV infection - chlorotic leaf margins, dark green streaks on petioles, narrow leaves that bunched at the top and severe stunting (Fig. 2). Total DNA was extracted from 25 leaf samples collected from plants with symptoms; they were then tested for BBTV by polymerase chain reaction (PCR) according to the published protocols. Two oligonucleotide primer pairs, mREP-F and mREP-R, specific for a ~240 bp conserved domain of BBTV DNA-mRep segment (Mansoor et al., 2005), and Scp-F and Scp-R specific for a ~1075 bp BBTV DNA-S that encodes coat protein gene (Amin et al., 2008) were used for PCR amplification. The amplicons of expected size were obtained from 23 of the 25 samples analysed suggesting the presence of BBTV in the affected plants (Fig. 3). The PCR products of DNA-mRep segment amplified from the BBTV-infected banana samples collected in Zoungue (GenBank Accession No. JQ437548) and Mitro (JQ437549) were sequenced in both directions directly from the purified PCR products. These two sequences showed 100% nucleotide sequence identity with a BBTV isolate from Cameroon (FJ580970) and 99-100% identity with several other BBTV isolates from the GenBank database belonging to the South Pacific group, comprised of BBTV isolates from Africa, Australia, India and South Pacific (Fig. 4). This finding confirmed that the virus isolate associated with the diseased plants in Benin indeed was of the BBTV South Pacific type. This finding constitutes the first report of BBTV in Benin. The disease is widespread in all the four communes surveyed (Fig. 1). Further surveys are necessary to assess the extent of BBTV occurrence in Benin. Until now, Cameroon was the western most frontier of BBTV presence in sub-Saharan Africa (Oben et al., 2009). This finding confirms spread of the disease into West Africa, and underscores an urgent need for intensive surveys in neighbouring countries and implementation of strict phytosanitary measures to prevent movement of planting material from the infected zones and to prevent further spread of BBTV.

Prevalence of Borrelia Burgdorferi Infection in a Series of 98 Primary Cutaneous Lymphomas

Borrelia burgdorferi has been variably associated with different forms of primary cutaneous lymphoma. Differences in prevalence rates among reported studies could be a result of geographic variability or heterogeneity in the molecular approaches that have been employed. In the present study, we investigated the prevalence of Borrelia burgdorferi sensu lato DNA in diagnostic tissue samples from fresh cutaneous biopsies of 98 primary cutaneous lymphomas and 19 normal skin controls. Three different polymerase chain reaction (PCR) protocols targeting the hbb, flagellin, and Osp-A genes were used. Direct sequencing of both sense and antisense strands of purified PCR products confirmed the specificity of the amplified fragments. Sequence specificity was assessed using the Basic Local Alignment Search Tool, and MultAlin software was used to investigate the heterogeneity of target gene sequences across the different samples. Borrelia DNA was not detected in 19 controls, 23 cases of follicular lymphoma, 31 cases of extranodal marginal zone lymphoma, or 30 cases of mycosis fungoides. A single case of 14 diffuse large B-cell lymphoma cases was positive for B. burgdorferi. This study does not support a pathogenic role of B. burgdorferi in primary cutaneous B- and T-cell lymphomas from areas nonendemic for this microorganism and the consequent rationale for the adoption of antibiotic therapy in these patients.

Evaluation of a SYTO9 real-time polymerase chain reaction assay to detect and identify pathogenic Leptospira species in kidney tissue and urine of New Zealand farmed deer

A SYTO9 real-time polymerase chain reaction assay for detection of pathogenic Leptospira spp. based on amplification of DNA gyrase subunit B (gyrB) gene has been optimized and evaluated for sensitivity and specificity on kidney and urine samples of New Zealand farmed deer. The detection limit was 10(3) cells/ml (2-10 copies/reaction). Comparison of the assay on deer kidneys (n = 268) with culture as the gold standard revealed a sensitivity and specificity of 85% and 99.2%, respectively. For deer urine (n = 113), the assay was compared with known inoculated samples and revealed a sensitivity and specificity of 96.7% and 100%, respectively. The assay was applied for quantifying pathogenic leptospires shed naturally in deer urine and revealed a detectable concentration of 3.7 × 10(3) to 1.7 × 10(6) cells/ml. To assess the assay's capability for identifying pathogenic Leptospira spp., 14 field isolates of L. borgpetersenii serovar Hardjo-bovis and L. interrogans serovar Pomona were amplified for polymerase chain reaction (PCR) product, purified, and sequenced. When compared with the National Center for Biotechnology Information database, sequence data matched with L. borgpetersenii serovar Hardjo-bovis in 13 samples and L. interrogans serovar Pomona in 1 sample, which was consistent with the microscopic agglutination test (MAT). Sequence analysis of purified PCR product amplified directly from kidney and urine samples also yielded serovar-comparable MAT results. Results suggest that the assay is rapid, sensitive, and specific for detection of pathogenic leptospires in deer clinical samples. The developed assay can also be used for estimating the concentration of leptospires and identifying Leptospira spp. in combination with DNA sequencing.

Mutations Found in the pncA Gene of Mycobacterium tuberculosis in Clinical Pyrazinamide-resistant Isolates from a Local Region of China

This study was designed to investigate the presence of mutations in the pncA gene, minimum inhibitory concentrations and pyrazinamidase activity of pyrazinamide-resistant Mycobacterium tuberculosis. In total, 47 M. tuberculosis clinical isolates from a local region of China were assayed. Pyrazinamidase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and a 721 bp region, including the entire pncA open-reading frame, 104 bp of the upstream sequence and 59 bp of the downstream sequence, was determined by DNA sequencing of purified polymerase chain reaction products. Of the 47 isolates resistant to pyrazinamide, 44 lost pyrazinamidase activity and had pncA mutations that occurred mainly near pyrazinamidase's active or metal ion binding sites; nine of them have not been reported previously. Three pyrazinamide-resistant isolates carried the wild-type pncA sequence and retained pyrazinamidase activity. These results show the molecular mechanism of pyrazinamide resistance in China and may also contribute towards the prevention of tuberculosis in China.

Presence of Helicobacter pylori in a Mexican Pre-Columbian Mummy

BackgroundRecent studies showed that Helicobacter pylori existed in the New World prior to the arrival of Columbus. The purpose of the present study was to detect the presence of Helicobacter pylori in pre-Columbian mummies from Northern Mexico.MethodsSix samples were studied (four samples of gastric remains, tongue-soft palate, and brain remained as negative controls) from two of the six naturally mummified corpses studied (adult male and infant male). Samples were taken from tissues suitable for DNA amplification by Polymerase chain reaction (PCR). DNA was extracted and H. pylori detection was carried out by PCR and hybridized with the pHp probe from 16S rRNA gene. The purified PCR products were cloned and sequenced in both directions. DNA sequences were analyzed with ALIGN and BLAST software. A second amplification was performed using ureB gene by real-time PCR.ResultsFrom four samples of gastric remnant, only two were H. pylori-positive for amplification of a 109 bp DNA fragment; the remaining two were negative, as were the tongue-soft palate and the brain biopsies as well. These PCR products were hybridized with a pHp probe. Nucleotide sequence analysis showed homology with H. pylori in 98 of 99% when compared with the gene bank nucleotide sequence. Only one sample of gastric remnant H. pylori-positive with 16S rRNA gene was also positive for ureB gene from H. pylori.ConclusionThis data supported infection with H. pylori in Mexican pre-Columbian mummies dating from approximately 1,350 AC.

Molecular analysis of sixteen unrelated factor XIIIA deficient families from south‐east of Iran

Molecular analysis of sixteen unrelated factor XIIIA deficient families from south‐east of Iran

Rapid identification of chicken anemia virus in Nigerian backyard chickens by polymerase chain reaction combined with restriction endonuclease analysis

Chicken infectious anemia (CIA) has not been routinely diagnosed in other avian species apart from commercial chickens. A sensitive polymerase chain reaction (PCR) combined with restriction endonuclease (RE) analysis was used for the detection and characterization of chicken anemia virus (CAV) in backyard chickens (Gallus gallus domesticus) in Nigeria. Using a pair of primers designed to amplify a 733 bp fragment in the VP1 (capsid protein) gene of CAV, the PCR assay detected CAV DNA in 9 of 12 serum samples from apparently healthy backyard chickens. RE digestion of the purified PCR products of commercial and backyard chickens with CfoI yielded two separate restriction endonuclease patterns, suggesting that Nigerian backyard chicken CAVs differed from the commercial chicken isolates at the nucleotide sequence level. Results of RE analysis also suggested that the backyard chickens contained a mixed population of CAV strains. These findings confirm that backyard chickens are susceptible to CAV infection and could also harbor the virus.

Rapid identification of chicken anemia virus in Nigerian backyard chickens by polymerase chain reaction combined with restriction endonuclease analysis

Chicken infectious anemia (CIA) has not been routinely diagnosed in other avian species apart from commercial chickens. A sensitive polymerase chain reaction (PCR) combined with restriction endonuclease (RE) analysis was used for the detection and characterization of chicken anemia virus (CAV) in backyard chickens (Gallus gallus domesticus) in Nigeria. Using a pair of primers designed to amplify a 733 bp fragment in the VP1 (capsid protein) gene of CAV, the PCR assay detected CAV DNA in 9 of 12 serum samples from apparently healthy backyard chickens. RE digestion of the purified PCR products of commercial and backyard chickens with CfoI yielded two separate restriction endonuclease patterns, suggesting that Nigerian backyard chicken CAVs differed from the commercial chicken isolates at the nucleotide sequence level. Results of RE analysis also suggested that the backyard chickens contained a mixed population of CAV strains. These findings confirm that backyard chickens are susceptible to CAV infection and could also harbor the virus.

BRAF Mutations in Juvenile Myelomonocytic Leukemia.

Approximately 75% of patients with juvenile myelomonocytic leukemia (JMML) harbour mutations in PTPN11, NF1 and RAS genes. The remaining cases presumably carry somatic mutations in other genes in the RAS pathway. BRAF plays a central role in this pathway between RAS and downstream molecules including MEK and ERK. BRAF mutations frequently occur in cancer. Recently, BRAF mutations were found in leukemia. Besides that, germline BRAF mutations cause cardio-facio-cutaneous syndrome, which shares many features with Noonan syndrome (NS). NS predisposes to a myeloproliferative disease resembling JMML. In 65 JMML patients screening for V600E mutations in exon 15 of the BRAF gene was performed from mononuclear cells. In a subset of patients, without RAS or PTPN11 mutations, and no clinical signs of NF1, the entire coding sequence of BRAF was analyzed. Sequence analysis was performed by direct, bidirectional sequencing of purified polymerase chain reaction products. In none of the 65 cases a V600E mutation of the BRAF gene was found. In a subset of patients in which the entire coding sequence of BRAF was analyzed, no mutations were identified either. Mutant proteins of the RAS-RAF-MEK-ERK pathway play an important role in the pathogenesis of JMML, resulting in GM-CSF hypersensitivity. In about 75% of the JMML cases these mutations affect RAS, NF1 or PTPN11 genes. The hypothesis for this study was that BRAF might play an important role in JMML as it is an important downstream effector of RAS. Our data show that apparently BRAF mutations do not play a role in JMML. Therefore, additional analysis of genes of the RAS pathway will be necessary to identify genetic aberrations in cases without known mutations.

Detection and Quantification of Y-Chromosomal Sequences by Real-Time PCR Using the LightCycler® System

Detection and Quantification of Y-Chromosomal Sequences by Real-Time PCR Using the LightCycler® System

Lack of Evidence for Prolonged Double-Long Terminal Repeat Episomal HIV DNA Stability In Vivo

Lack of Evidence for Prolonged Double-Long Terminal Repeat Episomal HIV DNA Stability In Vivo

Analysis of GATA-1 Mutations in Chronic Myeloid Leukaemia.

Despite extensive characterisation of the molecular pathogeneisis of chronic myeloid leukaemia (CML), little is known about the events underlying progression to accelerated phase and blast crisis. The haematopoietic transcription factor GATA1 plays an essential role in the commitment to, and differentiation within, erythroid, megakaryocyte, eosinophil and mast cell lineages. The observation of frequent eosinophilia and thrombocytosis alongside granulocytic hyperplasia suggests that GATA1 is actively expressed in CML chronic phase. GATA1 is mutated in the acute megakaryoblastic leukaemia of Down syndrome and in its preleukaemic form, transient abnormal myelopoiesis. The latter can morphologically resemble CML with basophilia and eosinophilia in addition to circulating blast cells.Examination of the expressed sequence tag (EST) database for GATA1 shows 4 variant sequences. 3 of these are derived from CML patient bone marrow.Given the plausible association between perturbations in GATA1 function and progression of CML, along with suggestive EST evidence, we hypothesized that mutations in GATA1 could be involved in the transition from chronic phase to blast crisis in CML.Materials and methods: Following appropriate informed consent and ethical approval genomic DNA was extracted from blast cells from 24 patients with CML in accelerated phase (n=1), myeloid blast crisis (n=15) or lymphoid blast crisis (n=8). Polymerase chain reaction (PCR) products were generated encompassing each coding exon of GATA1 and about 50 base pairs of flanking sequence. Exons 3 and 6 were divided into 2 fragments each for analysis. Gel purified PCR products were screened for mutations in GATA1 using DHPLC (denaturing high pressure liquid chromatography, WAVE, Transgenomics, USA). Samples with abnormal migration profiles were directly sequenced by bi-directional sequencing using an ABI 3100 Capillary Array sequencer (Perkin Elmer, UK). Mutations detected by this method were confirmed by a repeat PCR reaction, Topo cloning into pCR4TOPO vector (Invitrogen) and commercial sequencing (Agowa, Germany)Results and discussion 2/24 samples showed abnormal migration patterns by WAVE analysis. Sequencing confirmed 2 different single nucleotide changes in the intronic sequence flanking exon 5. These do not correspond to any known single nucleotide polymorphisms. Since these 2 mutations do not arise in coding or known regulatory regions of the gene it seems unlikely that they are of functional significance. Abnormal regulation of GATA1 activity either at the transcriptional or post-translational level may still play an important part in the pathogenesis of the disease. The mitogen-activated protein kinase (MAPK) pathway has recently been shown to be important in controlling the oncogenic potential of bcr-abl. MAPK mediated phosphorylation of GATA1 at critical serine residues promotes cell survival via increased Bcl-XL expression. In addition study of the upregulation of inducible heat shock protein 70 (Hsp70) in CML has shown the major p210 bcr-abl responsive element upstream of the Hsp70 promoter to be a GATA binding motif.Conclusion These findings suggest that GATA1 mutations are not frequent occurrences in the evolution of blast crisis from CML chronic phase. However, this study does not rule out an important role for dysregulated GATA1 activity in the molecular pathogenesis of chronic myeloid leukaemia.

A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis

A simple, two-step efficient method to perform multiple-site mutagenesis of a gene from bacterial genome was developed. The method was named polyacrylamide gel electrophoresis (PAGE)-mediated overlap extension polymerase chain reaction (PCR) (POEP). The first step involves synthesis of individual fragments containing mutant sites with 15- to 25-bp overlap between two adjacent fragments. Mutations were introduced into the overlapping oligonucleotide primers which ensured the particular primer-template annealing. PAGE was used to remove contaminating parental templates, mispriming fragments, and leftover primers. The second step involves synthesis of the mutant full-length fragment. All purified PCR products from the first step were combined and used as the template for a second PCR using high-fidelity DNA polymerase, with the two outermost flanking oligonucleotides as primers. Using the POEP method, we have successfully introduced eight EcoRI sites into the Escherichia coli beta-galactosidase (Lac Z) gene. The overall rate of obtaining the multiple mutant sites was 100%. The POEP method is simple, involving only two steps, and reliable for multiple-site mutagenesis and is promising to be widely used in gene modification.

Gynecological tumors revealing hereditary nonpolyposis colorectal cancer: analysis of a large Lebanese pedigree

The objective of this study was to evaluate the aggregation of colorectal cancer (CRC) and hereditary nonpolyposis colorectal cancer (HNPCC)–related extracolonic cancers in an extended Lebanese family with HNPCC. This was a pedigree analysis and a prospective follow-up over an 8-year period. The causative germ line mutation was detected using denaturing high-performance liquid chromatography, polymerase chain reaction (PCR) of short fluorescent fragments, and direct DNA sequencing of purified PCR products. The penetrance of CRC is high and accounts for approximately two thirds of risk carriers with an early age of onset (21 years). The extracolonic cancer spectrum includes ovary, endometrium, small bowel, skin, and brain, with an age of onset as early as 30 years. The causative mismatch repair gene mutation is an MSH2 point mutation involving the splice donor site of intron 3 (G→A). Scrutinized in genomic DNA from 35 consented members, it was found in 18 of them and cosegregates with the cancer phenotype in the family. Early-onset ovarian and endometrial carcinomas may reveal HNPCC families in the Middle Eastern region, with MSH2 germ line mutation. We propose a biannual screening program, starting around the age of 20–25 years, pending additional data on this topic.

Gynecological tumors revealing hereditary nonpolyposis colorectal cancer: analysis of a large Lebanese pedigree

The objective of this study was to evaluate the aggregation of colorectal cancer (CRC) and hereditary nonpolyposis colorectal cancer (HNPCC)-related extracolonic cancers in an extended Lebanese family with HNPCC. This was a pedigree analysis and a prospective follow-up over an 8-year period. The causative germ line mutation was detected using denaturing high-performance liquid chromatography, polymerase chain reaction (PCR) of short fluorescent fragments, and direct DNA sequencing of purified PCR products. The penetrance of CRC is high and accounts for approximately two thirds of risk carriers with an early age of onset (21 years). The extracolonic cancer spectrum includes ovary, endometrium, small bowel, skin, and brain, with an age of onset as early as 30 years. The causative mismatch repair gene mutation is an MSH2 point mutation involving the splice donor site of intron 3 (G-->A). Scrutinized in genomic DNA from 35 consented members, it was found in 18 of them and cosegregates with the cancer phenotype in the family. Early-onset ovarian and endometrial carcinomas may reveal HNPCC families in the Middle Eastern region, with MSH2 germ line mutation. We propose a biannual screening program, starting around the age of 20-25 years, pending additional data on this topic.

Linkage of ciprofloxacin resistance with a single genotypic cluster of Klebsiella pneumoniae

The objective of this study was to examine the epidemiology of ciprofloxacin-resistant, extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae strains. Sixty-nine unique patient isolates of K. pneumoniae isolated from a variety of clinical specimens submitted to the clinical bacteriology laboratories of The Royal Infirmary of Edinburgh and associated General Practices were identified and susceptibility testing was performed with the Vitek system. Strains flagged as ESBL-positive by the Vitek system were subjected to isoelectric focusing. The results suggested that all 69 isolates harboured at least one ESBL, which was later confirmed by polymerase chain reaction (PCR) with bla TEM and/or bla SHV primers. The purified PCR product was subjected to automated sequencing and the results were compared with the BLAST online search engine. Of the 69 isolates, 32 (46.4%) were found to be resistant to ciprofloxacin, 11 (15.9%) were intermediate and 26 (37.7%) were sensitive. To investigate the epidemiological relationship between the ciprofloxacin-resistant ESBL-positive strains, pulsed-field gel electrophoresis (PFGE) was performed. Rapidest software was used to calculate the genetic distance by the Nei distance method. PFGE analysis indicated that the clinical isolates belonged to four distinct genotype clusters (Groups A, B, C and D); each group or cluster was homogeneous or compact with respect to certain characteristics. Group A consisted of 25 isolates, group B of 3 isolates and Groups C and D of 2 isolates each. These results indicate that the spread of resistance is largely as a result of the dissemination of a single clonal strain. PCR was used to amplify the gyrA and parC genes from genomic DNA of the ciprofloxacin-resistant isolates. The amplified product was sent for analysis by automated DNA sequencing and the resulting DNA sequences were compared with the gyrA gene of K. pneumoniae. The sequencing results demonstrated that alteration of the GyrA subunit of DNA gyrase at amino acid 83 and/or amino acid 87 plays a central role in conferring high-level quinolone resistance in K. pneumoniae possessing ESBLs.

Characteristics of the beta myosin heavy chain gene Ala26Val mutation in a Chinese family with hypertrophic cardiomyopathy

Background Genotype–phenotype studies have suggested that some mutations of genes encoding various components of the cardiac sarcomere cause hypertrophic cardiomyopathy (HCM) and are associated with the prognosis of patients with HCM. The aims of this study were to investigate the gene mutations of exons in the cardiac beta myosin heavy chain (MYH7) gene, the troponin T (TNNT2) gene, and the brain natriuretic peptide (BNP) gene, as well as to assess the effect of these mutations on the clinical features of Chinese patients with HCM. Methods Five unrelated Chinese families with HCM were studied. Exons 3 and 18 in the MYH7 gene, exon 9 in the TNNT2 gene, and all three exons in the BNP gene were screened with the polymerase chain reaction (PCR) for genomic DNA amplification. Further study included purification of PCR products and direct sequencing of PCR fragments by fluorescent end labeling. Results A C-to-T transition in codon 26 of exon 3 in the MYH7 gene was found in one family (including four patients and five carriers), resulting in an amino acid substitution of valine (Val) for alanine (Ala). The Ala26Val mutation was of incomplete dominance (penetrance 44%). This mutation was not seen in the other four families or in the control group. Moreover, the association between the gene mutations of exon 18 in MYH7, of exon 9 of TNNT2, and of all three exons in BNP and HCM was not found in the populations we studied. Conclusions The missense mutation Ala26Val found in this one Chinese family was associated with a mild phenotype of HCM. The genetic and phenotypic heterogeneity of HCM exists in the Chinese population. It suggests that genetic and environmental factors may be involved in the pathogenesis of HCM.

Williamsia muralisPulmonary Infection

<i>Williamsia muralis</i>Pulmonary Infection

Demonstration That Mast Cells, T Cells, and B Cells Bearing the Activating Kit Mutation D816V Occur in Clusters within the Marrow of Patients with Mastocytosis

Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering.

SIRPH analysis: SNuPE with IP-RP-HPLC for quantitative measurements of DNA methylation at specific CpG sites.

This chapter describes a detailed protocol using single-nucleotide primer extension (SNuPE) for quantitative analysis of DNA methylation on specific CpG sites. The first step DNA sample to be studied is treated with sodium bisulfite, which converts selectively unmethylated cytosines to uracil, while methylated cytosines remain unconverted. Subsequently, a SNuPE reaction is performed, with an oligo just flanking a CpG site, using a purified polymerase chain reaction product derived from bisulfite-treated DNA as a template. The oligo is extended by either ddCTP or ddTTP depending on whether the site is methylated or unmethylated, respectively. The reaction is quantitative and linear, and two to three sites can be studied simultaneously in a multiple reaction. The SNuPE product, without further purification, is separated by ion-pair reverse-phase (IP RP) high-performance liquid chromatography (using an alkylated nonporous polysterene-divinylbenzene cartridge) that allows an easy, semiautomated method for separation of the extended and unextended products and an accurate quantification of the extended products. The ratio of the ddCTP to the ddTTP gives the fraction of the methylated cytosines at that specific CpG site.