The following are examples of peer-reviewed studies in which TaKaRa Ex Taq or Titanium Taq DNA Polymerase were used to amplify DNA sequences that are present in low- or single-copy in samples for a variety of biomedical research aims:
Watanabe, K. et al. (2011) A novel method for ABO genotyping using a DNA chip. J. Forensic Sci.56:S183–S187.
Summary: ABO blood type genotyping remains a useful technique for some forensic applications such as identifying individuals using highly degraded DNA samples from decomposed tissue. The authors developed a method for ABO genotyping that involves PCR amplification of portions of the ABO and D17Z1 genes using TaKaRa Ex Taq HS DNA Polymerase and subsequent hybridization of the amplified products to a chip bearing fluorescently-labeled probes corresponding to known SNPs. ABO genotypes could be determined quickly by assessing the fluorescence intensity of each probe. A 2013 article described use of the method for highly degraded DNA samples, reporting accurate results with 0.5 ng template DNA.
Summary: Hepatitis B virus replicates though an RNA intermediate generated by its non-proofreading reverse transcriptase, resulting in tremendous genetic diversity. Mutant subpopulations may therefore co-exist with wild-type HBV within a single host, complicating detection and treatment. To understand the prevalence and clinical relevance of HBV mutants in chronically infected adults, the authors sequenced HBV DNA isolated from serum samples from 192 patients. Researchers focused on specific mutations in the basal core promoter (BCP), which controls replication of HBV genomic DNA, and the precore (preC) region, which encodes secreting hepatitis B e antigen (HBeAg) - an antigen that contributes to HBV persistence in the host. TaKaRa Ex Taq polymerase was used for two rounds of nested PCR designed to detect BCP A1762T/G1764A mutations (which affect HBV replication initiation) and the preC G1896A mutation (which results in truncated preC protein, thereby abolishing production of HBeAg). A1762T/G1764A mutations were more common in genotype C HBV than in genotype B HBV and correlated with a lower HBsAg level in patient serum samples.
Summary: Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), although the role of specific HPV mutations on the development of HCC is unclear. The authors performed nested PCR and sequencing of HBV DNA in samples derived from serum of 135 patients with HPV-related HCC, focusing on mutations in the S region (which encodes envelope protein) and the basal core promoter (BCP). TaKaRa Ex Taq DNA Polymerase was used for PCR. Mutations in the BCP region were not correlated with tumor characteristics or patient survival rate. In contrast, a significantly lower survival rate was found for patients infected with HBV with mutation in the pre-S region. Deletion mutations in the pre-S region leads to accumulation and aggregation of large envelope proteins.
Summary: Human papillomavirus (HPV) infection is correlated with cervical cancer (CC), but there are additional risk factors for cancer development. The authors assessed polymorphic mitochondrial DNA mutations in HPV-positive and HPV-negative CC cases. Because mitochondrial dysfunction and the presence of homoplastic somatic mutations in mtDNA had been documented previously for other types of cancer, they hypothesized that mtDNA mutations play a role in HPV-related CC. A 1.1-kb fragment of the mtDNA D-loop region, which controls mtDNA replication and transcription, was amplified using TaKaRa Ex Taq polymerase and sequenced to assess mtDNA polymorphisms. One mutation, C150T, was positively associated with HPV infection and subsequent CC risk among Chinese women.
Summary: The authors analyzed transmission of several commensal viruses during blood transfusions using matched donor and serial recipient blood samples. Viruses analyzed included non-enveloped DNA anellovirus species TTV, TTMV, and TTMDV, which are highly genetically diverse and prevalent in humans, and the enveloped RNA virus GVB-C. Two rounds of PCR were performed to amplify anellovirus DNA from serum samples. Similarly, two rounds of PCR were used to amplify a portion of the GVB-C 5' UTR from cDNA prepared from serum. TaKaRa Ex Taq DNA Polymerase was used for all amplifications. Products were purified and sequenced to impute donor-to-recipient transmission. Transmission of GBV-C through blood transfusion was detected in one of three informative donor-recipient pairs, but the transient nature of GBV-C infection complicated detection. For anelloviruses, widespread pre-existing infection was found in both donors and recipients and one incidence of TTMDV transmission from donor to recipient was confirmed. This superinfection involved transmission of TTMDV from the donor to a recipient who already harbored a genetically distinct TTMDV infection.
Summary: For unknown reasons, approximately 1 in 300 individuals infected with HIV-1 maintain plasma viral load at undetectable levels (<50 viral RNA copies per ml), even without antiviral therapy. Such individuals are referred to as “elite controllers” (EC). Theories about why EC can maintain low viral load include host genetics, host innate and adaptive immune responses, and/or viral sequence variations. In this study, researchers analyzed viral sequence variations in 95 HIV-1 EC subjects. TaKaRa Ex Taq polymerase was used for the second round of PCR to amplify viral and proviral products prior to sequencing. Analysis of viral genetic defects, viral length polymorphisms, and codon-by-codon comparisons of HIV-1 from EC versus chronic progressor (CP) patients indicated no differences. This led researchers to conclude that spontaneous control of HIV replication in ECs is not attributable to shared viral genetic defects or shared viral polymorphisms. The authors noted how critical PCR success was, stating that the “generation and analysis of viral sequences derived from HIV-1 EC represents a substantial challenge, since PCR amplification from such small quantities of starting material is problematic. Despite these difficulties, we were able to obtain viral genetic information from 90% of EC subjects analyzed, including 75% of EC plasma samples.”
Summary: Japanese physicians reported the case of a 1-year-old boy with an infected subdural hematoma, caused by hematogenous Streptococcus pneumoniae infection of a pre-existing, asymptomatic, chronic subdural hematoma. Identification of S. pneumoniae was performed by latex agglutination testing and by PCR amplification of the S. pneumoniae lytA gene using TaKaRa Ex Taq DNA Polymerase.
Summary: Approximately 90% of adults are infected with Jamestown Canyon virus (JCV), which remains quiescent in kidney and lymphoid tissue during latency but can be activated under immunosuppressive conditions. JCV is an oncovirus and various studies have linked it to colorectal, prostate and esophageal cancers, brain tumors, bronchopulmonary carcinoma, and B cell lymphoma. Researchers assessed techniques to identify JCV in DNA extracted from lung and gastric carcinomas, normal lung tissues, normal gastric mucosa, and tonsils. Nested PCR with TaKaRa Ex Taq HS DNA Polymerase was used to amplify the T-antigen, agnoprotein, and VP genes. The amplified products were then analyzed by Southern blotting and sequencing. Real-time PCR, immunohistochemistry, in-situ hybridization, and in-situ PCR were also assessed as detection techniques. The JCV targets were readily amplified from DNA extracted from lung and gastric carcinomas, leading authors to recommend nested PCR in combination with Southern blot and sequencing as a comparatively sensitive approach for detection of JCV genomic DNA in human non-neural tissues, in contrast to immunological techniques.
Summary: Researchers developed a semi-quantitative multiplex PCR assay for simultaneous detection of Bacillus anthracis, Yersinia pestis, and Leishmania species in blood samples. In proof-of-concept experiments, total DNA from cultured B. anthracis (Sterne), Y. pseudotuberculosis, Leishmania species, and T. brucei was isolated and used to spike human whole blood to determine the limit of detection of the assay. Multiplex PCR was performed using TaKaRa Ex Taq polymerase and SYBR® Green I, and melt-derivative analysis was performed. Additional experiments used blood samples from 11 visceral leishmaniasis patients. The assay enabled DNA detection in blood spiked with < 50 target cells/ml for all pathogens tested, and was 100% sensitive for the detection of L. donovani from leishmaniasis patients.
Summary: Lipophilic yeast of the genus Malassezia, which are inherently difficult to culture and study in laboratory settings, colonize skin of the head, neck, and shoulders and can exacerbate atopic dermatitis (AD). The authors used a nested PCR assay with TaKaRa Ex Taq polymerase to analyze DNA extracted from topical dressings applied to the skin of AD patients or healthy controls. Using purified DNA, the limit of detection of the assay was determined to be 1 fg DNA. Malassezia-specific DNA was detected in 100% of samples obtained from 32 AD patients and 78% of healthy subjects, with a greater species diversity observed in AD patients. Nested PCR was recommended as a valuable technique for analyzing Malassezia spp. as an alternative to fungal culturing attempts.
Summary: The authors describe a technique for estimating SNP allele frequency in pooled DNA populations. The method, named alternately binding probe competitive PCR (ABC-PCR), uses PCR for competitive amplification of two alleles in presence of a probe that hybridizes next to the SNP site. The probe is labeled with BODIPY FL (green) at the 5' end and TAMRA (red) at the 3' end. Green fluorescence is quenched if an adjacent guanine residue is present; therefore, endpoint measurement of green fluorescence intensity indicates the ratio of the two SNP alleles, while endpoint measurement of red fluorescence intensity indicates bound versus unbound probe. (The technique is not applicable to A/T substitutions.) In proof-of-concept experiments, allele frequencies of ALDH2, GNB3, and HTR2A were estimated by ABC-PCR for defined DNA mixtures of PCR-amplified alleles or pools of human genomic DNA. Estimated allele frequencies strongly correlated to expected ratios for all three SNPs with high accuracy and with acceptable relative standard deviation when allele frequencies exceeded 5%. Titanium Taq polymerase was used for amplifications.
Summary: Tumors frequently exhibit changes in chromosomal copy number due to general karyotype instability as well as changes in regions particularly susceptible to copy number aberrations (CNAs) and loss of heterozygosity (LOH). While previous studies had reported use of comparative genomic hybridization and genotype mapping arrays to detect changes in tumor sample chromosomal copy number by assessing the relative strength of genomic signal, such studies assumed a predominantly diploid chromosomal background - which is frequently not the case for tumors. The authors analyzed CNAs present in glioblastoma multiforme (GBM) tumors using Affymetrix 500K Mapping Arrays. Titanium Taq polymerase was used for all PCR amplifications. Researchers found that many or most of the chromosomes in 24 GBH tumor samples were, in fact, aneuploid.
Summary:The range of HIV-1 genotypes within a patient can affect response to various antiviral drugs. For example, the antiviral agents abacavir (ABC) or didanosine (ddI) select for strains with a K65R or L74V mutation in HIV-1 reverse transcriptase. The authors developed an allele-specific (AS) real-time PCR assay using Titanium Taq polymerase to detect low-level K65R mutations in HIV-1 viral cDNA prepared from patient plasma. The AS-PCR assay was used to assess baseline K65R levels in 63 treatment-naïve patients and to monitor K65R mutation levels in 154 treatment-experienced patients. Researchers found that prior treatment with ABC or ddI was associated with a significantly higher incidence of detectable K65R mutations. Further treatment with the antiviral drug tenofovir disoproxil fumarate (TDF) was associated with poor virological response to therapy and expansion of preexisting K65R mutant population.
Summary: Gastrointestinal tuberculosis (TB) is difficult to diagnose using the routine approaches of endoscopy and biopsy or surgical biopsy. The authors used a PCR assay to amplify the IS6110 element of Mycobacterium tuberculosis from fecal samples of patients with intestinal TB or healthy controls. The IS6110 element is specific to M. tuberculosis and contains an internal SalI restriction site, allowing confirmation of the PCR product by digestion. Titanium Taq polymerase was used for PCR. The sensitivity and specificity of the PCR assay for identification of intestinal TB was 88.8% and 100%, respectively—an improvement over histopathology (sensitivity of 50%) and culture methods (sensitivity of 33%).
Summary: The authors compared two methods for whole genome amplification prior to SNP analysis. The first method, OmniPlex technology (Rubicon Genomics), used Titanium Taq polymerase for PCR amplification of libraries with universal primers. The other method investigated was multiple strand displacement, which uses a phi29 polymerase and random hexamers in an isothermal reaction. Both methods were found to perform exceptionally well for amplifying DNA for SNP genotyping, with 99.9% concordance in genotyping results between unamplified and amplified samples. The authors noted that the OmniPlex method “…has the advantage that it enables the creation of whole-genome DNA libraries from degraded as well as intact DNA samples…[which] can be archived and repeatedly reamplified.”
Summary: The authors investigated whether RNA could be recovered from dried blood samples stored on filter paper since 1975 and used for RT-PCR analysis. After RNA extraction and cDNA preparation from samples that had been stored for 1 month, 21 years, or 27 years, Titanium Taq polymerase was used to amplify human beta-actin (205 bp fragment) or GAPDH (97 bp fragment). Amplification was successful for all samples tested.