OOCH25˜OH3˜3˜3˜OH allows strand extension at 3˜ end normal deoxyribonucleoside triphosphate (dNTP) OOCH25˜3˜ H prevents strand extension at 3˜end chain-terminating dideoxyribonucleoside triphosphate (ddNTP) basebase PPP PPP ATGTCAGTCCAG  3˜RESULT TACAGTCAGGTC  5˜GCAT ATG GCAT ATGTCAG GCAT ATGTCAGTCCAG G sequence of DNA primer sequence read from gel 3˜  CGTATACAGTCAGGTC  5˜3˜  CGTATACAGTCAGGTC  5˜5˜  GCAT  3˜GCAT A GCAT ATGTCA GCAT ATGTCAGTCCA GCAT AT GCAT ATGT GCAT ATGTCAGT GCAT ATGTC GCAT ATGTCAGTC GCAT ATGTCAGTCC A T C G A C C T G A C T G T A A T C G 3˜5˜ADD LABELED DNA PRIMER single-stranded DNA fragment to be sequenced T T T TTT T T T T A A A A AA AA GG GGG GG CC C C C CC C CADD DNA POLYMERASE AND DIVIDE INTO 4 SEPARATE TUBES ADD EXCESS AMOUNTS OF NORMAL dNTPs sequence of original DNA strand ADD SMALL AMOUNT OF ONE CHAIN-TERMINATING ddNTP TO EACH TUBE TT C T A T AG T G T C A CC T AAATA G C TT GG C G T AATC A T GG T(B) (A) GCATATG GCATA GCATATGT GCATAT GCATATGTC mixture of DNA products, each containing a chain-terminating ddNTP labeled with a different fuorescent marker PRODUCTS LOADED ONTO CAPILLARY GEL electrophoresis size-separated products are read in sequence Dideoxy sequencing, or Sanger sequencing (named after the scientist who invented it), uses DNA polymerase, along with special chain-terminating nucleotides called dideoxyribonucleoside triphosphates (left), to make partial copies of the DNA fragment to be sequenced. These ddNTPs are derivatives of the normal deoxyribonucleoside triphosphates that lack the 3˜ hydroxyl group. When incorporated into a growing DNA strand, they block further elongation of that strand. DNA SEQUENCING MANUAL DIDEOXY SEQUENCING AUTOMATED DIDEOXY SEQUENCING To determine the complete sequence of a single-stranded fragment of DNA (gray), the DNA is frst hybridized with a short DNA primer (orange) that is labeled with a fuorescent dye or radioisotope. DNA polymerase and an excess of all four normal deoxyribonucleoside triphosphates (blue A, C, G, or T) are added to the primed DNA, which is then divided into four reaction tubes. Each of these tubes receives a small amount of a single chain-terminating dideoxyribonucleoside triphosphate (red A, C, G, or T). Because these will be incorporated only occasionally, each reaction produces a set of DNA copies that terminate at different points in the sequence. The products of these four reactions are separated by electrophoresis in four parallel lanes of a polyacrylamide gel (labeled here A, T, C, and G). In each lane, the bands represent fragments that have terminated at a given nucleotide but at different positions in the DNA. By reading off the bands in order, starting at the bottom of the gel and reading across all lanes, the DNA sequence of the newly synthesized strand can be determined (see Figure 8–25C). The sequence, which is given in the green arrow to the right of the gel, is complementary to the sequence of the original gray single-stranded DNA. Fully automated machines can run dideoxy sequencing reactions. (A) The automated method uses an excess amount of normal dNTPs plus a mixture of four different chain-terminating ddNTPs, each of which is labeled with a fuorescent tag of a different color. The reaction products are loaded onto a long, thin capillary gel and separated by electrophoresis. A camera (not shown) reads the color of each band as it moves through the gel and feeds the data to a computer that assembles the sequence. (B) A tiny part of the data from such an automated sequencing run. Each colored peak represents a nucleotide in the DNA sequence. 5˜  GCAT 3˜  CGTA 479SEQUENCING WHOLE GENOMES Shotgun sequencing: To determine the nucleotide sequence of a whole genome, the genomic DNA is frst fragmented into small pieces and a genomic library is constructed, typically using plasmids and bacteria (see Figure 8–30). In shotgun sequencing, the nucleotide sequence of tens of thousands of individual clones is determined; the full genome sequence is then reconstructed by stitching together (in silico) the nucleotide sequence of each clone, using the overlaps between clones as a guide. The shotgun method works well for small genomes (such as those of viruses and bacteria) that lack repetitive DNA. SECOND-GENERATION SEQUENCING TECHNOLOGIES The dideoxy method made it possible to sequence the genomes of humans and most of the other organisms discussed in this book. But newer methods, developed since 2005, have made genome sequencing even more rapid—and very much cheaper. With these so-called second-generation sequencing methods, the cost of sequencing DNA has decreased dramatically. Not surprisingly, the number of genomes that have been sequenced has increased enormously. These rapid methods allow multiple genomes to be sequenced in parallel in a matter of weeks, enabling investigators to examine thousands of individual human genomes, catalog the variation in nucleotide sequences from people around the world, and uncover the mutations that increase the risk of various diseases, from cancer to autism. These methods have also made it possible to determine the genome sequence of extinct species, including Neanderthal man and the wooly mammoth. By sequencing genomes from many closely related species, they have also made it possible to understand the molecular basis of key evolutionary events in the tree of life, such as the “inventions” of multicellularity, vision, and language. The ability to rapidly sequence DNA has had major impacts on all branches of biology and medicine; it is almost impossible to imagine where we would be without it. BAC clones: Most plant and animal genomes are large (often over 109 nucleotide pairs) and contain extensive amounts of repetitive DNA spread throughout the genome. Because a nucleotide sequence of a fragment of repetitive DNA will “overlap” every instance of the repeated DNA, it is diffcult, if not impossible, to assemble the fragments into a unique order solely by the shotgun method. To circumvent this problem, the human genome was frst broken down into very large DNA fragments (each approximately 100,000 nucleotide pairs) and cloned into BACs (see p. 469). The order of the BACs along a chromosome was determined by comparing the pattern of restriction enzyme cleavage sites in a given BAC clone with that of the whole genome. In this way, a given BAC clone can be mapped, say, to the left arm of human Chromosome 3. Once a collection of BAC clones was Thousands of genomes from individual humans have now been sequenced and it is not necessary to painstakingly reconstruct the order of DNA sequence “reads” each time; they are simply assembled using the order determined from the original human genome sequencing project. For this reason, resequencing, the term applied when the genome of a species is sequenced again (even though it may be from a different individual), is far easier than the original sequencing. sequence one strand of fragments GTTCAGCATTG------GCCATTAGTTCA---GCCATTAGTTCAGCATTG---original sequence reconstructed based on sequence overlap sequences of two fragments AA D B BA B C E C restriction pattern for individual BAC clones restriction map of one segment of human genome cleavage sites for restriction nucleases A, B, C, D, and E obtained that spanned the entire genome, each individual BAC was sequenced by the shotgun method. At the end, the sequences of all the BAC inserts were stitched together using the knowledge of the position of each BAC insert in the human genome. In all, approximately 30,000 BAC clones were sequenced to complete the human genome. random fragmentation multiple copies of genome 479