THE EFFECT OF COUPLING EFFICIENCY ON THE YIELD AND PURITY OF OLIGONUCLEOTIDES SYNTHESIZED USING PHOSPHORAMIDITE METHOD.
Journal Title: WORLD JOURNAL PHARMACY AND PHARMACEUTICAL SCIENCE - Year 2017, Vol 6, Issue 12
Abstract
Coupling efficiency is a way of measuring how efficiently the DNA synthesizer is adding new bases to the growing DNA chain. To measure coupling efficiency, dimethyl trityl (DMT) group is used, which is colorless when attached to DNA base, but gives a characteristic orange color once removed upon addition of the base to the growing chain of DNA. The intensity of this color can be measured by UV spectrometry and it is directly related to the number of DMT molecules released throughout the synthesis. The aim of this study was to determine the effect of coupling efficiency on the yield and purity of the synthesized oligonucleotides by using the DNA synthesizer Mermade 12. Brucella-specific primers Forward (5’CATGCGCTATGTCTGGTTTAC3’) reverse (5’TAATAAGACTCGGCTTTGTGA3’), were arranged in the synthesis chamber with CPG standard columns C and A, respectively, with an estimated activator volume of 3.320 ml, trityl average size 10, trityl area threshold 20000. β-actin primers, forward (5’GGACTTCGAGCAAGAGATGG3’) reverse (5’AGCACTGTGTTGGCGTACAG3’), were arranged in the synthesis chamber with CPG standards columns G and G, respectively and an estimated activator volume 3.230 ml, trityl average size 10, trityl area threshold 20,000. The purity and quantity of the primers was determined by Epoch microplate spectrophotometer. Brucella-specific forward and reverse primers showed average coupling efficiencies of 99.97 and 99.95%, with a purity (OD260/280) of 1.85 and 1.84 and yields of 2456ng/μl and 2440 ng/μl, respectively. The β-actin forward and reverse primers showed average coupling efficiencies of 68% and 42%, with purities of 1.14 and 1.12 and yields of 468 and 365 ng/μl, respectively. The coupling efficiency correlates with quantity and purity of synthesized oligonucleotides. Hence, oligonucleotides with low coupling efficiency should not be used in downstream experiments.
Authors and Affiliations
Kandanath B. M.
Keywords
Related Articles
- EP ID EP619184
- DOI -
- Views 150
- Downloads 0
CONCEPTUAL STUDY OF BHRAJAK PITTA W.S.R. TO SHVITRA
Ayurveda is an ancient health science which helps in the eradication of disease as well as maintaining health of healthy person.It forms a precious base of Indian culture. It is best among all the health sciences because...
ANTIMICROBIAL AND PHYTOCHEMICAL STUDIES OF CARICA PAPAYA LEAVES AGAINST ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS.
The bioactive compound of Carica papaya leaves were extracted using methanol and hexane. They were investigated for their antibacterial activity against some selected organism, Escherichia coli and Staphylococcus aureus....
PREPARATION AND EVALUATION OF PERINDOPRIL CONTROLLED RELEASE MATRIX TABLETS USING DIFFERENT POLYMERS LIKE HPMC K100M, GUM CARRAGEENAN AND PECTIN.
Perindopril is an Angiotensin-Converting Enzyme (ACE) inhibitor, used to Hypertension (high blood pressure) and Congestive Heart Failure (CHF). These inhibitors relax (arterioles) the muscles around small arteries. They...
EFFECT OF CINNAMON SUPPLEMENTATION ON GUT MICROBIOTA, SATIETY HORMONAL AND LIVER FUNCTION AMONG SAMPLE DIABETIC OBESE PATIENTS
This study was planned to investigate the effects of cinnamon supplementation on gut flora levels, satiety hormone and liver function among type2 diabetic obese patients. Methods and Patients: 30 obese subjects with type...
DETERMINATION OF TOTAL CONCENTRATION OF PROTEINS AND CARBOHYDRATES IN TAPEWORM POSTGANGESIA ARMATA AND INTESTINES OF INFECTED AND NON-INFECTED FISH HOST SILURUS GLANIS
The study investigated the concentration of some biochemical parameters in the tissues of the Postgangesia armata and intestine of their final host (Fish) Silurus glanis. The study showed significant increases(P0.05) in...