Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 67
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To obtain the sequence of a long stretch of DNA, a set of overlapping fragmentsis prepared, the sequence of each is determined, and all sequences are then combined. The procedures arestraightforward, and DNA sequences have accumulated at such aPage 211rapid rate that large computer databases are necessary to manage the hundreds of millions of nucleotides of DNAsequence already determined for a variety of genes from many organisms.
A still larger database would be requiredto handle the DNA in human sperm or eggs, which consists of 3 × 109 nucleotide pairs. Although the sequencingtechnique described here is the manual method still in use in many research laboratories, in Chapter 9 we will seehow DNA sequencing can be automated and how DNA-sequencing machines work.The dideoxy sequencing method employs DNA synthesis in the presence of small amounts of nucleotides thatcontain the sugar dideoxyribose instead of deoxyribose (Figure 5.38). Dideoxyribose lacks the 3'-OH group, whichis essential for attachment of the next nucleotide in a growing DNA strand, so incorporation of a dideoxynucleotideinstead of a deoxynucleotide immediately terminates further synthesis of the strand.
To sequence a DNA strand,four DNA synthesis reactions are carried out. Each reaction contains the single-stranded DNA template to besequenced, a single oligonucleotide primer complementary to a stretch of the template strand, all fourdeoxyribonucleoside triphosphates, and a small amount of one of the nucleoside triphosphates in the dideoxy form.Each reaction produces a set of fragments that terminates at the point at which a dideoxynucleotide was randomlyincorporated in place of the normal deoxynucleotide. Therefore, in each of the four reactions, the lengths of thefragments are determined by the positions in the daughter strand at which the particular dideoxynucleotide presentin that reaction was incorporated.
The sizes of the fragments produced by chain termination are determined by gelelectrophoresis, and the base sequence is then determined by the following rule:If a fragment containing n nucleotides is generated in the reaction containing a particulardideoxynucleotide, then position n in the daughter strand is occupied by the base present in thedideoxynucleotide. The numbering is from the 5' nucleotide of the primer.For example, if a 93-base fragment is present in the reaction containing the dideoxyFigure 5.38Structures of normal deoxyribose and the dideoxyribose sugar used inDNA sequencing.
The dideoxyribose has a hydrogen atom (red) attachedto the 3' carbon, in contrast with the hydroxyl group (red) at this positionin deoxyribose. Because the 3' hydroxyl group is essential for theattachment of the next nucleotide in line in a growing DNA strand, theincorporation of a dideoxynucleotide immediately terminates synthesis.form of dATP, then the 93rd base in the daughter strand produced by DNA synthesis must be an adenine (A).Because most native duplex DNA molecules consist of complementary strands, it does not matter whether thesequence of the template strand or that of the daughter strand is determined.
The sequence of the template strandcan be deduced from the daughter strand because their nucleotide sequences are complementary. However, inpractice, both strands of a molecule are usually sequenced independently and compared in order to eliminate mostof the errors that can be made by misreading the gels.With short, single-stranded fragments, molecules that differ in size by a single base can be separated byelectrophoresis in an acrylamide gel. For example, if a reaction mixture contains a set of DNA fragments consistingof 20, 21, 22, .
. ., 100 nucleotides, then electrophoresis will yield separate bands—one containing the fragment ofsize 20, the next the fragment of size 21, and so forth. This extraordinary sensitivity to size is the basis of the DNAsequencing procedure.Page 212The Sequencing ProcedureThe procedure for sequencing a DNA fragment is diagrammed in Figure 5.39. In this example, the primer length is20 nucleotides. Termination at G produces fragments of 21, 24, 27, and 29 nucleotides; termination at A producesfragments of 22 and 30 nucleotides; termination at T produces a fragment of 26 nucleotides; and termination at Cproduces fragments of 23, 25, and 28 nucleotides.
Typically, either the primer or one of the deoxynucleotides usedcontains a radioactive atom, so that after electrophoresis to separate the fragments by size, the fragments can bevisualized as bands on photographic film placed over the gel. After electrophoresis and autoradiography, thepositions of the bases in the daughter strand can be read directly from the gel, starting with position 21, asGACGCTGCGA. This corresponds to the template strand sequence CTGCGACGCT.Figure 5.40 is an autoradiogram of an actual sequencing gel. The shortest fragments are those that move the fastestand farthest.
Each fragment contains the primer fragment at the 5' end of the daughter strand. The sequence can bereadFigure 5.39Dideoxy method o DNA sequencing. Four DN synthesis reactions arecarried out in the presence of al normal nucleotides plus a small amountof one of the dideoxynucleotides containing G, A, T, C. Synthesis continuesalong th template strand until a dideoxynucleotide is incorporated.The products that result from termination at each dideoxynucleotideare indicated at the right. The fragments are separated by size byelectrophoresis, and the positions of the nucleotides are determineddirectly from the gel.
In this example, the length of the primer neededto initiate DNA synthesis is 20 nucleotides. Lengths of the DNA fragmentsare shown at the left of the gel. The sequence of the daughter strand isread from the bottom of the ge as 5'-GACGCTGCGA-3'Page 213directly from the bottom to the top of the gel. The sequence of the first 16 bases in the segment in this gel, readfrom bottom to top, is5'-CACTGCCTGCGCCCAG-3'and so forth.Clinical Use of Dideoxynucleoside AnalogsOur knowledge of DNA structure and replication has applications not only in procedures for the manipulation ofDNA but also in the development of drugs for clinical use.
An example is in one approach to the treatment ofAIDS. A number of dideoxynucleoside analogs are effective in inhibiting replication of the viral genetic material.A few of these are illustrated in Figure 5.41. Recall that a nucleoside is a base attached to a sugar without aphosphate.
A nucleoside analog is a molecule similar, but not identical, in structure to a nucleoside. In Figure 5.41,ddC is the normal dideoxyribocytidine nucleoside. It is effective against AIDS, as are the dideoxynucleosideanalogs AZT, D4T, and ddI (and other such analogs). The nucleoside, rather than the nucleotide, is used in therapybecause the nucleotide, having a highly charged phosphate group, cannot cross the cell membrane as easily.
Theemergence of these drugs from our basic knowledge of DNA structure and replication is one of the prime examplesof the fact that "pure" science may have many unforeseen practicalFigure 5.40A section of a dideoxy sequencing gel.The sequence is read from the bottomto the top. Each horizontal rowrepresents a single nucleotide position inthe DNA strand synthesized from thetemplate. The vertical columns resultfrom termination by the dideoxyforms of G, A, T, or C. The sequencefrom the lower part of the gel isindicated.applications.
The basic experiments on DNA were carried out long before the recognition of AIDS as a distinctinfectious disease and the discovery that HIV is the causative agent.Figure 5.41A few of the drugs that have been found to be effective in the treatment of AIDS by interfering with the replication ofHIV virus. The technical names of the substances are as follows: ddI is 2',3'-dideoxyinosine; ddC is 2',3'-dideoxycytidine;AZT is 3'-azido-2',3'-dideoxythymidine; and D4T is 2',3'-didehydro-2',3'-dideoxythymidine.Page 214Chapter SummaryDNA is a double-stranded polymer consisting of deoxyribonucleotides. A nucleotide has three components: a base,a sugar (deoxyribose in DNA, ribose in RNA), and a phosphate.
Sugars and phosphates alternate in forming asingle polynucleotide chain with one terminal 3'-OH group and one terminal 5'-P group. In double-stranded(duplex) DNA, the two strands are antiparallel: Each end of the double helix carries a terminal 3'-OH group in onestrand and a terminal 5'-P group in the other strand. Four bases are found in DNA: adenine (A) and guanine (G),which are purines; and cytosine (C) and thymine (T), which are pyrimidines.
Equal numbers of purines andpyrimidines are found in double-stranded DNA, because the bases are paired as A–T pairs and G–C pairs(Chargaff's rules). This pairing holds the two polynucleotide strands together in a double helix. The basecomposition of DNA varies from one organism to the next. The information content of a DNA molecule resides inthe sequence of bases along the chain, and each gene consists of a unique sequence.The double helix replicates by using enzymes called DNA polymerases, but many other proteins also are needed.Replication is semiconservative in that each parental single strand, called a template strand, is found in one of thedouble-stranded progeny molecules.
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