PCR primer design for detection of SNPs in SLC22A1 rs683369 encoding OCT1 as the main transporter of metformin
Elida Zairina, Junaidi Khotib, Chrismawan Ardianto, Syed Azhar Syed Sulaiman, Charles D. Sands, Timothy E. Welty in Unity in Diversity and the Standardisation of Clinical Pharmacy Services, 2017
Polymerase Chain Reaction (PCR) is a method widely applied in molecular biology as it can amplify a target DNA sequence quickly, easily, and inexpensively, generating millions of copies. PCR consists of three main stages, namely denaturation, annealing, and extension, that run consecutively to generate target DNA strands exponentially (Joshi and Deshpande 2010), (Gabriyan and Avashia 2013). A supply of nucleotide base (dNTP), Taq polymerase enzyme, and primer pair is required to amplify DNA molecules. A primer is a single short strand of DNA that co-owns a complementary sequence with DNA template and is important as a marker of polymerase enzyme to begin an amplification process, so it must have the ability to bind specifically to the target sequence (Borah 2011). Therefore, a specific primer design and optimum PCR condition are crucial to DNA sequence replication for polymorphism analysis. This study aimed to obtain a specific primer and optimum PCR condition for amplification of DNA sequence containing SLC22A1 rs683369 that encodes OCT1.
Genetic and genomic investigations
Angus Clarke, Alex Murray, Julian Sampson in Harper's Practical Genetic Counselling, 2019
Genetic laboratories have used several methods to determine the DNA sequence of specific genes. Most have depended upon the PCR to amplify the specific fragment of DNA that is of interest, from among the rest of the patient's DNA obtained from their blood or other tissues. DNA primers are constructed to flank the stretches of sequence in which mutations are often found and then these are amplified using PCR to generate a sufficient quantity of ‘pure’ (or at least ‘clean’) material for analysis. Amplification of DNA by cloning can also be used. The DNA from one or a few PCRs will then be sequenced by the conventional (Sanger) method. The amplified DNA is copied by a DNA polymerase in four separate reactions with a small proportion of dideoxynucleotides (ddNTPs) present in the standard mix of nucleotides, and with a fluorescent label attached to one of the four ddNTPs. When a ddNTP is incorporated, that molecule can no longer be extended and so the length of the molecule with the ddNTP at its end identifies the nucleotide at that specific position in the gene.
Biology of microbes
Philip A. Geis in Cosmetic Microbiology, 2006
To understand the process in detail requires far more effort. In reality, at least seven enzymes are involved in the process described in the paragraph above: initiator protein, helicase, polymerases, repair nucleases, topoisomerase, single-strand DNA-binding proteins, and DNA ligase. The initiator protein first finds the right place to begin copying and guides the helicase to the correct position (an origin of replication site) on the nucleic acid. The helicase separates the DNA by breaking the weak bonds between the nucleotides to unwind the two strands of DNA. Then the polymerases arrive to join the free nucleotides to their matching complements on the old strands using the phosphate bond energy from the nucleotide to help form the new bond to the other nucleotides as they are added to the existing chain. These polymerases work along with primases that first synthesize a short (one to five nucleotides long) RNA primer. This primer allows DNA polymerase to begin catalyzing the addition of nucleotides to a new strand complementary to the existing template upon which the new DNA synthesis is based.
Real-Time Multiplex PCR Analysis in Infectious Uveitis
Published in Seminars in Ophthalmology, 2019
Caroline L. Minkus, Paulo J. M. Bispo, George N. Papaliodis, Lucia Sobrin
Since its first description in 1985, polymerase chain reaction (PCR) has been used to rapidly and accurately amplify a given stretch of DNA available in a sample.11,14 First, the sample is heated to a temperature at which the hydrogen bonds that hold the two complimentary strands of DNA together are broken, allowing the DNA to denature and the two strands to separate. A predetermined DNA oligonucleotide is added, and the temperature lowered to allow primer annealing. The primer serves as the foundation block for a new strand of DNA, which a DNA polymerase builds once the mixture is at a suitable temperature for DNA synthesis. This cycle of heating to denature DNA, and cooling to allow for doubling of the amount of DNA in the sample, is repeated until the target molecule has been adequately amplified.15 Previous modifications to standard PCR have used shared wells with primers for multiple DNA targets present in the same reaction. This technique, multiplex PCR, has the benefit of requiring a smaller initial sample to amplify several DNA targets.14,16
Anti-neuroinflammatory effects of cudraflavanone A isolated from the chloroform fraction of Cudrania tricuspidata root bark
Published in Pharmaceutical Biology, 2018
Kwan-Woo Kim, Tran Hong Quang, Wonmin Ko, Dong-Cheol Kim, Chi-Su Yoon, Hyuncheol Oh, Youn-Chul Kim
Total RNA was isolated from the cells using Trizol (Invitrogen, Carlsbad, CA), according to the manufacturer’s recommendations, and spectrophotometrically quantified (at 260 nm wavelength). Total RNA (1 mg) was reverse transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, CA). The cDNA was amplified with SYBR Premix Ex Taq kit (TaKaRa Bio, Shiga, Japan) using a StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA). Briefly, each 20 mL of the reaction mixture contained 10 mL of SYBR Green PCR Master Mix, 0.8 mM of each primer, and diethyl pyrocarbonate (DEPC) treated water. The primer sequences were designed using PrimerQuest (Integrated DNA Technologies, Cambridge, MA). The sequences of primers used in this experiment are shown in Table 1. The optimum conditions for PCR amplification of cDNA were established by following the manufacturer’s instructions. The data were analysed using StepOne software (Applied Biosystems, Foster City, CA) and the cycle number at the linear amplification threshold (Ct) values for the endogenous control gene (GAPDH) and the target gene was recorded. Relative gene expression (target gene expression normalized to the expression of the endogenous control gene) was calculated by using the comparative Ct method (2–ΔΔCt). The analysis was conducted three times, independently.
An integrative understanding of the large metabolic shifts induced by antibiotics in critical illness
Published in Gut Microbes, 2021
Andrea Marfil-Sánchez, Lu Zhang, Pol Alonso-Pernas, Mohammad Mirhakkak, Melinda Mueller, Bastian Seelbinder, Yueqiong Ni, Rakesh Santhanam, Anne Busch, Christine Beemelmanns, Maria Ermolaeva, Michael Bauer, Gianni Panagiotou
The concentration of genomic DNA was determined by Qubit, and the DNA quality was checked on the gel. 200 ng of DNA was used as input for PCR reaction with corresponding primer set specifically binding to different hypervariable regions. Each primer set had a unique barcode. PCR product was then run on the gel and DNA fragment with the proper amplification size was cut and purified. The purified PCR product was then used as template for library preparation. The PCR products were pooled together with equal amount and then end polished, A-tailed, and ligated with the adapter. These fragments were filtered with beads again. After PCR reaction (to make library fully double strand), the library was analyzed for size distribution and quantified using real-time PCR. The library was then to be sequenced on Hiseq2500.
Related Knowledge Centers
- Biochemistry
- Directionality
- DNA
- DNA Polymerase
- DNA Replication
- In Vitro
- Molecular Biology
- Nucleic Acid
- Nucleotide
- Oligonucleotide Synthesis