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DNA SEQUENCING

Sequencing involves determination of the base sequence of DNA, in this method the single stranded DNA to be sequenced is used as template for DNA synthesis by DNA polymerase, a radiolabeled primer complementary to 3’ end of target DNA is added along with the four deoxynucleoside triphosphates, the sample is divided in four reaction tubes, and a small amount of one of the four dideoxy nucleoside triphosphates linked to florescent dyes are added to each tube In first reaction tube adenosine dideoxynucleotide triphosphate (ddATP) which lacks 3’ and 2’ hydroxyl groups is added in little amount. (click) The ddATP can be inserted into the growing chain of DNA ( click) but after its insertion further elongation cannot take place and elongation is stopped. ( click), as a result different sized DNA fragments are produced In second reaction tub, cytidine dideoxynucleotide triphosphate (ddCTP) lacking 3’ and 2’ hydroxyl groups is added in little amount. (click) The ddCTP can be inserted into the growing chain of DNA ( click) but after its insertion further elongation cannot take place and elongation is stopped. ( click), as a result DNA fragments of varying lengths are produced In third reaction tube, guanosine dideoxynucleotide triphosphate (ddGTP) deficient in 3’ and 2’ hydroxyl groups is added in little amount. (click) The ddGTP can be inserted into the growing chain of DNA ( click) but after its insertion further elongation cannot take place and elongation is stopped. ( click), as a result DNA fragments of varying lengths are produced In fourth reaction tube,thymidine dideoxynucleotide triphosphate (ddTTP)is added in little amount. (click) The ddTTP can be inserted into the growing chain of DNA ( click) but after its insertion further elongation cannot take place and elongation is stopped. ( click), as a result DNA fragments of varying lengths are produced The pieces of newly synthesized DNA in each tube are separated by capillary electrophoresis in a different lane of the gel. The sequence of the new strand can be read from the smallest to the largest fragments ( click )on the gel, e.g., from the bottom to the top or 5’ to 3’ end. (click) So reading the sequence from bottom to top makes it CTTGGAACTGTA, C corresponds to 10 nucleotide fragment T corresponds to end of 11 nucleotide fragment, T at end of 12 nucleotide fragment, G at ends of 13 and 14 nucleotide fragments consecutively, followed by adenines at ends of 15 and 16 nucleotide fragments, a cytosine at end of 17 nucleotide fragment, thymine at end of 18 nucleotide fragment, G at end of 19 nucleotide fragment, T at end of 20 nucleotide fragment and A at end of 21st nucleotide fragment Normally The 3’ hydroxyl group of the deoxy ribose of one nucleotide to the 5’ hydroxyl group of an adjacent nucleotide through a phosphoryl group. In ddNTPs both 2’ and 3’ ends are unavailable therefore the chain terminates when these are inserted The main limitation of sanger dideoxy sequencing method is speed. Improvements in this technique have led to next generation sequencing which allows whole genome to be sequenced in one day. It involves Mechanical fractionation of genome followed by Rapid sequencing and alignment Many segments sequenced simultaneously The sequences are read from bottom to top of gel, for normal it makes AATATCATCTTTGGT , for Cystic fibrosis it can be read as AATATCATTGGTGTT in this case the sequences for control and cystic fibrosis are identical for first eight bases. Positions 12 to 15 of normal gene and 9 to 12 of CF gene are identical therefore there is a three base deletion in CF gene corresponding to bases 9 to 11 of normal gene since it is a deletion of three bases there some researchers do not classify it as frameshift mutation. The DNA pattern is indicative of deletion of codon leading to absence of phenylalanine at 508th position in CFTR Summary: • Four test tubes: • A sample of DNA to be sequenced • DNA polymerase • deoxyribonucleotide triphosphates • In each tube one of four dideoxynucleotide triphosphate (ddNTP) which lacks 3’ and 2’ hydroxyl groups is added. • The ddNTP will be inserted into the growing chain of DNA after its insertion elongation is stopped. • The pieces of newly synthesized DNA in each tube are separated by gel electrophoresis in separate lanes of the gel. • The sequence of the new strand can be read from the smallest to the largest fragments on the gel, e.g., from the bottom to the top.