DNA Sequencing PDF

DNA sequencing Ohoud M. Marie MD, PhD – Biochemistry Bioinformatics  Why DNA sequencing ?? The definitive understanding of a DNA molecule is derived from knowledge of its nucleotide sequence. The function of a gene can often be deduced by comparing a determined, but...

DNA sequencing Ohoud M. Marie MD, PhD – Biochemistry Bioinformatics  Why DNA sequencing ?? The definitive understanding of a DNA molecule is derived from knowledge of its nucleotide sequence. The function of a gene can often be deduced by comparing a determined, but unknown, sequence with sequences of genes with known functions. In addition, nucleotide sequence information is important for determining the presence of single base pair mutations.  DNA sequencing The most commonly used DNA sequencing procedure is the dideoxynucleotide method developed by F. Sanger (b. 1918), who received Nobel Prizes for inventing sequencing techniques for both protein and DNA. A dideoxynucleotide is a human-made molecule that lacks a hydroxyl group at both the 2’- and 3’-carbons of the sugar moiety  DNA sequencing During DNA replication, an incoming natural nucleoside triphosphate, which is determined by base pairing to the nucleotide of the template strand, is linked by its 5’ a-phosphate group to the 3’-hydroxyl of the last nucleotide of the growing chain. However, if a dideoxynucleotide is incorporated at the end of the growing chain, DNA synthesis stops, because a phosphodiester bond cannot be formed with the next incoming nucleotide  DNA sequencing The first step in the standard laboratory procedure for dideoxynucleotide DNA sequencing entails annealing a synthetic oligonucleotide (primer) to a predetermined segment of a strand of the cloning vector near the insertion site of the cloned DNA. A primer, which provides a 3’-hydroxyl group for the initiation of DNA synthesis, is usually from 17 to 24 bases long to ensure that it base pairs to a specific complementary sequence. Shorter primers might base pair to more than one sequence in the DNA construct, which would produce multiple initiation sites. Of course, the 3’-hydroxyl group of the primer “points” in the direction of the DNA to be sequenced.  DNA sequencing The primed DNA sample is partitioned into four separate reaction tubes. Each tube contains four deoxyribonucleotides (dATP, dCTP, dGTP, and dTTP), one of which is radiolabeled; one of the four dideoxynucleotides (ddATP, ddCTP, ddGTP, or ddTTP); and DNA polymerase. The concentration of each dideoxynucleotide in each reaction tube is carefully adjusted to ensure that it is incorporated into growing chains at every possible site and not just at the first occurrence of the complementary nucleotide of the template strand.  DNA sequencing As a consequence of this important feature, after enzymatic DNA synthesis, each reaction tube will contain a unique set of oligonucleotides, each with a primer sequence.  DNA sequencing The synthesis of DNA is stopped by the addition of formamide, which also prevents the strands from forming base pairs. The contents of each tube are loaded into a well of a polyacrylamide gel, and the DNA molecules are separated by electrophoresis.  DNA sequencing This separation procedure resolves pieces of DNA that differ in size by as little as a single nucleotide. An autoradiograph of the gel shows only the radiolabeled DNA fragments produced during the enzymatic DNA synthesis step, with each lane corresponding to a reaction tube that contained one of the four dideoxynucleotides. The sequence of nucleotides of a segment of one strand of a cloned piece of DNA is determined by noting the order of the bands, as accurately as possible, from the bottom to the top of the autoradiograph.  DNA sequencing The fastest migrating band in the gel (the bottom-most radiolabeled fragment), which corresponds to the smallest DNA fragment. Between 250 and 350 bands can be resolved clearly on most autoradiographs. Usually, the primer sequence is positioned about 10 to 20 nucleotides away from the insertion site of the cloned DNA, so that a known sequence can be recognized at the start of reading of the autoradiograph, thereby identifying precisely the first nucleotide of the cloned DNA.  DNA sequencing Currently, the dideoxynucleotide method forms the basis of automated DNA sequencing. Sequence analysis can be carried out with four different fluorescent dyes, one for each dideoxynucleotidereaction. With a four-fluorescent dye system, the samples at the completion of each reaction are pooled and the fragments are separated in a single lane of a polyacrylamide gel. This type of analysis is called “4-color, 1-lane” detection.  DNA sequencing The emission data are recorded and stored in a computer. After a run is completed, the succession of fluorescent signals is converted to nucleotide sequence information.  DNA sequencing Generally, automated DNA sequencing systems read with high accuracy about 500 bases per run and, under optimal conditions, one instrument can resolve about 20,000 bases per hour.