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2015, Number 1

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Rev Cubana Hematol Inmunol Hemoter 2015; 31 (1)

Optimization of deoxyribonucleic acid extraction for molecular typing of human leukocyte antigens

Chang MA, Morera BLM, Ustariz GCR, Bencomo HA

Language: Spanish
References: 16
Page: 59-64
PDF size: 116.59 Kb.


ABSTRACT

For typing the Human Leukocyte Antigens (HLA) by Polymerase Chain Reaction using the Specific Sequence Primer (SSP) method, between 3 and 6 µg of high pureness Deoxyribonucleic Acid (DNA) are needed. In order to establish the best conditions for DNA extraction, as well as the optimal samples, an investigation was carried out in the Center for Cellular Engineering and Organs and Tissues Transplantations in Havana, Cuba. DNA was extracted from fresh and frozen samples of blood, buffy coat, coagulated blood and plasma from 15 volunteers and also extracted from a lymphocyte suspension. A “QIAcube” extractor anda Kit “QIAamp DNA Blood Mini” were used. The DNA concentration and pureness was measured by an “EPOCH” microdot spectrophotometer running “Gen 5” software. From 200 µL of blood or buffy coat means of 5.8 and 22.4 µg of DNA, respectively, were obtained and in all cases the amount of DNA was over 3 µg. One hundred percent of plasma samples and the 6.6 % of coagulated blood samples yielded less than 3 µg of DNA. Forty µg of DNA were extracted from a cell suspension containing 30 × 106 lymphocytes. The A260/A280 ratio was between 1.7 and 2 in all eluates. The fresh or frozen blood and buffy coat yielded an optimum amount of DNA in the experiments, but not in plasma nor in coagulated blood. The highest amount of DNA was extracted from a lymphocyte suspension. All eluates were of fine pureness.


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