Teknik Long Polymerase Chain Reaction (LPCR) Untuk Perbanyakan Kerangka Baca Terbuka Gen Pengkode Polimerase Virus Hepatitis B

Teknik PCR dapat dikembangkan untuk mengamplifikasi potongan DNA dengan ukuran panjang. Salah satu teknik yang digunakan adalah Long PCR (LPCR). Teknik LPCR sering digunakan untuk mengamplifikasi gen atau potongan DNA yang berukuran lebih panjang ketika PCR standar tidak dapat diaplikasikan untuk amplifikasi tersebut. Tujuan dari penelitian ini adalah untuk memperoleh kerangka baca terbuka gen pengkode polimerase virus hepatitis B (PolHBV) menggunakan metode LPCR, dan menentukan urutan nukleotida potongan DNA tersebut. Amplifikasi dilakukan menggunakan Taq polimerase. Kerangka baca terbuka gen pengkode PolHBV telah berhasil diamplifikasi dengan LPCR yang telah dimodifikasi. Hal tersebut diindikasikan dengan keberadaan pita DNA berukuran pasangan basa (bp) pada elektroforesis gel agarosa yang mendekati ukuran teoritisnya yaitu 2532 bp. Hasil penentuan urutan nukleotida parsial menunjukkan bahwa potongan DNA yang diperoleh memiliki homologi 98% dengan gen PolHBV yang dideposit di GenBank.Technique of PCR can be improved to permit the amplification of longer DNA fragment. One of this technique is Long PCR (LPCR). LPCR is often used to amplify larger genes or large segment of DNA which standard PCR is not applicable. The objective of this study is to obtain the open reading frame of gene encoding polymerase of HBV (polHBV) using LPCR method, and to determine the nucleotide sequence of DNA fragment encoding polHBV. The amplification was conducted using Taq polymerase. The open reading frame of gene encoding polHBV was successfully amplified using modified LPCR method. It was identified by a band between 2000 dan 3000 base pairs (bp) DNA marker. The determination of nucleotide sequence informed that DNA fragment obtained was 98% homologue to the gene encoding PolHBV deposited on GenBank.

Optimasi Pcr (Polymerase Chain Reaction) Fragmen 724 Pb Gen Katg Multi Drug Resistance Tuberculosis Untuk Meningkatkan Produk Amplifikasi

Deteksi adanya mutasi pada gen katG MDR-TB (Multi Drug Resistance Tuberculosis) yang bertanggung jawab terhadap resistensi isoniazid (INH) dapat dilakukan dengan teknik Polymerase Chain Reaction (PCR). Pada penelitian ini metode PCR digunakan untuk mengamplifikasi fragmen berukuran 724 pb gen katG. Telah dilakukan percobaan pendahuluan, di mana proses PCR berhasil mengamplifikasi fragmen berukuran 724 pb namun masih menghasilkan pita yang sangat tipis yang menunjukkan bahwa proses amplifikasi belum optimal. Oleh sebab itu, penelitian ini bertujuan untuk mengoptimasi proses PCR agar mampu meningkatkan produk amplifikasi sehingga diperoleh pita yang tebal. Produk PCR yang tebal ini cukup memadai untuk proses sekuensing. Tahap optimasi yang dilakukan dalam proses PCR meliputi penambahan jumlah templat DNA pada formula PCR, variasi suhu annealing, penambahan waktu annealing dan waktu ekstensi. Hasil optimasi menunjukkan penambahan jumlah templat DNA menjadi 1 µL, suhu annealing 56ºC, waktu annealing 1 menit 20 detik, dan waktu ekstensi 2 menit memberikan amplifikasi terbaik karena menghasilkan pita yang tebal dan tidak terjadi mispriming

Microfluidic polymerase chain reaction

We implement microfluidic technology to miniaturize a thermal cycling system for amplifying DNA fragments. By using a microfluidic thermal heat exchanger to cool a Peltier junction, we have demonstrated rapid heating and cooling of small volumes of solution. We use a miniature K-type thermocouple to provide a means for in situ sensing of the temperature inside the microrefrigeration system. By combining the thermocouple, two power supplies controlled by a relay system, and computer automation, we reproduce the function of a commercial polymerase chain reaction thermal cycler and demonstrate amplification of a DNA sample of about 1000 base pairs

The polymerase chain reaction

The Polymerase Chain Reaction (PCR) is a technique for the in vitro synthesis of billions of copies of a specific nucleic acid sequence by performing successive rounds of in vitro nucleic acid replication. This is achieved by using two oligonucleotide primers that hybridize (annealing) to the opposite strand of the target DNA at positions that flank the region to be amplified through simultaneous extension of both primers

Effectiveness of rotavirus vaccination in prevention of hospital admissions for rotavirus gastroenteritis among young children in Belgium : case-control study

Objective : To evaluate the effectiveness of rotavirus vaccination among young children in Belgium. Design : Prospective case-control study. Setting : Random sample of 39 Belgian hospitals, February 2008 to June 2010. Participants : 215 children admitted to hospital with rotavirus gastroenteritis confirmed by polymerase chain reaction and 276 age and hospital matched controls. All children were of an eligible age to have received rotavirus vaccination (that is, born after 1 October 2006 and aged >= 14 weeks). Main outcome measure : Vaccination status of children admitted to hospital with rotavirus gastroenteritis and matched controls. Results : 99 children (48%) admitted with rotavirus gastroenteritis and 244 (91%) controls had received at least one dose of any rotavirus vaccine (P= 12 months. The G2P[4] genotype accounted for 52% of cases confirmed by polymerase chain reaction with eligible matched controls. Vaccine effectiveness was 85% (64% to 94%) against G2P[4] and 95% (78% to 99%) against G1P[8]. In 25% of cases confirmed by polymerase chain reaction with eligible matched controls, there was reported co-infection with adenovirus, astrovirus and/or norovirus. Vaccine effectiveness against co-infected cases was 86% (52% to 96%). Effectiveness of at least one dose of any rotavirus vaccine (intention to vaccinate analysis) was 91% (82% to 95%). Conclusions : Rotavirus vaccination is effective for the prevention of admission to hospital for rotavirus gastroenteritis among young children in Belgium, despite the high prevalence of G2P[4] and viral co-infection

Polymerase chain reaction in clinical practice

One of the most heralded developments in basic science to reach clinical application in recent years has been the Polymerase Chain Reaction (PCR). PCR has been applied in various areas of clinical medicine including rapid diagnosis of viral, bacterial, fungal and parasitic disease, the diagnosis and prediction of inherited disease, the detection of an association between certain viruses and specific cancers, the detection of organ transplant rejection and HLA subtyping. In basic research PCR is useful in identification of point mutation, deletion, insertions, rearrangements, amplifications and translocations

Efficiency of polymerase chain reaction processes: A stochastic model

A stochastic model for the efficiency of polymerase chain reaction (PCR) processes is presented. The model is based on the assumption that the i’th nucleotide incorporation on the DNA template takes τi seconds, where τi has an exponential distribution with mean 1/λi. In this paper, given λi that can be obtained from the primer-template sequence, temperature profile, enzyme rate, and other assay conditions [3], we calculate the efficiency of a multi-step PCR process using the distribution of the summation of non-identical exponential distributions