D1S80 locus is highly polymorphic and has been used worldwide as an important marker for forensic, medical analysis and paternity tests. Vent DNA Polymerase is a high-fidelity thermophilic DNA polymerase. The fidelity of Vent DNA polymerase is 5 to 15 folds higher than that observed for Tag DNA polymerase. This high fidelity derives in part from an integral 3’-5’ proofreading exonuclease activity in Vent DNA polymerase. Greater than 90% of the polymerase activity remains after one hour incubation at 95oC. Because of these advantages, we want to apply Vent DNA polymerase for the amplification of D1S80 alleles by PCR. Our study showed that PCR condition for Vent DNA polymerase are rather different than for Tag DNA polymerase. The amplification of D1S80 alleles with Vent DNA polymerase is optimal with the following parameters: 1. A final concentration of MgSO4 in PCR mixture of 4 mM. 2. An annealing temperature for the specific D1S80 primers 68oC. 3. A final concentration of dNTPs of 200mcM.
DNA polymerase ; Alleles

No abstract available.
DNA* ; Polymerase Chain Reaction*

Study on blood samples of 29 patients with hemophilia A and 25 normal people to determine the mutation of intron 18 and 19 of factor VIII by PCR-RFLP using Vent DNA polymerase assay. PCR and RFLP methods were used. Results: 16/29 (55%) of haemophilia A patients has the mutation in intron 18 of factor VIII gene and none of them has the mutation in intron 19. This PCR-RFLP assay can be applied for detection of mutations and these methods can be used for carrier analysis and prenatal diagnosis of hemophilia A.
Hemophilia A ; Polymerase Chain Reaction ; DNA

No abstract available.
DNA* ; Hemophilia A* ; Polymerase Chain Reaction*

No abstract available.
Diagnosis* ; DNA* ; Polymerase Chain Reaction*

No abstract available.
DNA* ; Humans* ; Polymerase Chain Reaction*

The nature of hepatitis B virus (HBV) particle associated DNA polymerase was studied in relation to various enzyme inhibitors including antiviral agents. HBV DNA polymerase required high concentration of MgCl2(> 30 mM) and neutral pH for its full activity. p-chloromercuribenzoate was a strong inhibitor (85% inhibition at 1 mM) but N-ethylmaleimide had much less inhibitory effect (20% inhibition at 10 mM). Phosphonoformic acid showed the greatest inhibitory effect on HBV-DNA polymerase (almost complete inhibition at 100 microM) among phosphocompounds tested. Adenine arabinoside triphosphate (ara-ATP) and cytosine arabinoside triphosphate (ara-CTP) were competitive inhibitors with respect to their respective deoxyribonucleoside triphosphate (dATP and dCTP). Ara-CPT was a stronger inhibitor of HBV-DNA polymerase compared to ara-ATP. Ki values for ara-ATP and ara-CTP were 15.0 microM and 11.7 microM , respectively. HBV-DNA polymerase is characteristic in its ionic requirements and susceptibilities to certain inhibitors.
DNA-Directed DNA Polymerase/antagonists & inhibitors ; DNA-Directed DNA Polymerase/metabolism* ; Hepatitis B Virus/enzymology* ; Human

Beta-Thalassemia is a blood disease with beta-globin deficiency caused by a directly down-regulation in the synthesis of structurally normal beta chains. Vent DNA polymerase is a high-fidelity thermophilic DNA polymerase, and the fidelity 5 - 15 times higher than Taq DNA polymerase (1/31,000 vs. 1/290 - 1/2,400). Using ARMS PCR (Amplification Refractor Mutation System-PCR) with Vent DNA polymerase, we found that 9/28 (32%) of the tested beta-Thalassemia patients had the mutation at codon 17 and 4/28 (14%) at codon 41/42. This method can be applied for a rapid prenatal diagnosis of beta-Thalassemia disease and has public health significance.
beta-Thalassemia ; DNA ; Polymerase Chain Reaction ; Diagnosis

Deep vent DNA polymerase is purified from a strain of E. Coli that carries the Deep Vent DNA polymerase gene from pyrococcus species GB-D. The native organism was isolated from a submarine thermal vent at 2010 meters and is able to grow at temperature as high as 104oC. Deep Vent DNA polymerase is a high-fidelity thermophilic DNA polymerase. The Deep Vent DNA polymerase is derived from an integral 3'-5' proofreading exonuclease activity. Deep Vent DNA polymerase is ven more stable than Vent DNA polymerase at temperature of 95 to 100oC. Because of these advantages, we want to apply Deep Vent DNA polymerase for amplification of NS4 gene - hepatitis C virus by PCR. Our study showed that PCR conditions for Deep Vent DNA polymerase are rather different than for Taq DNA polymerase. The amplification of NS4 gene - hepatitis C virus with Deep Vent DNA polymerase is optimal with the following parameters: (1) A final concentration of MgSO4 in PCR mixture of 4 mM. (2) An annealing temperature of 57oC. (3) A final concentration of dNTPs of 200 mM.
Hepatitis C ; Deep Vent DNA polymerase

For highly degraded DNA samples of forensic casework, new miniSTR PCR systems have been developed to supplement the current CODIS STRs. In the present study, we established the three miniplexes for nine miniSTRs (NC01 : D10S1248, D14S1434 and D22S1045; NC02 : D1S1677, D2S441 and D4S2364; and NC03 : D3S3053, D6S474 and D20S482) which had been previously suggested by Butler group (NIST, Gaitherburg, MD, USA). To evaluate the usefulness of the nine miniSTRs in analysis of degraded DNA, the sensitivity and efficacy of the three miniplexes were determined and then compared with those of the BigMini STR system which consists of six CODIS miniSTRs (TH01, CSF1PO, FGA, TPOX, D7S820, and D21S11). The three miniplexes gave better results in both the sensitivity test and efficiency test in comparison with BigMini. In the sensitivity test using serially diluted standard DNA, most loci in the three miniplexes showed reliable results for samples containing 50 pg of DNA and some even showed good sensitivity for samples containing 30 pg of DNA. Additionally, the three miniplexes generated useful profiles for both enzymatically degraded DNA and 50-year old skeletal remain samples. Among the nine miniSTRs, D4S2364, D3S3053, D14S1434, and D1S1677 produced the most successful DNA profiles for old skeletal remains. These results suggest that new miniSTRs could be useful supplements to the 13 CODIS STRs for forensic analysis of degraded DNA.
DNA* ; Humans ; Middle Aged ; Polymerase Chain Reaction