Gene Cloning

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Sequencing DNA 197 company (Celera) attempted to sequence the genome using a wholegenome shotgun approach. The company invested in a large number of sequencing machines and high-powered computers that would be essential for the assembly. The prospect of sequencing the human genome, at least to draft level, using the whole-genome shotgun approach only became feasible once sequencing technology and computing power had advanced to the stage it was at in the late 1990s. Celera constructed libraries of 2, 10 and 50 kb fragments from five individuals. They sequenced both ends of clones from the three different libraries. Remarkably, Celera completed their part of the sequencing phase of the project in under a year. However, they did not obtain enough data from their own reads to complete the assembly. Instead they exploited the sequence information that had been made available by the publicly funded project. This data they shredded into 550 bp reads and added to their own sequencing reads. In total they obtained 27.27 million reads from their own sequencing reads and 16.06 million ‘reads’ from the publicly funded project, the combined set giving eight-fold coverage of the genome. Celera then looked for sequence overlap between the 43.33 million reads to assemble the sequence into contigs essentially as described in Figure 7.8. Because they had sequenced from both ends of the clones, especially the 10 and 50 kb clones, it was possible to arrange the sequence contigs into scaffolds (Figure 7.9). The chromosomal positions of the sequence contigs and scaffolds were then determined by exploiting STS marker information and the BAC fingerprint information. Celera published their draft whole-genome shotgun assembly at the same time as the publicly funded project published their draft sequence. The Celera draft assembly contained over 220,000 contigs with over 220,000 gaps. Like the publicly funded draft sequence it included 90% of the euchromatic DNA. The Celera publication did contain a slightly better assembly, which they termed a compartmentalized shotgun assembly, which had exploited positional information from a variety of sources including the publicly funded project. It is clear that the quality of the whole-genome shotgun assembly does not match the quality of the hierarchical shotgun assembly. One of the reasons the publicly funded project had opted for the hierarchical approach was that, although it was agreed that both approaches would yield a large amount of draft sequence information very quickly, the hierarchical approach was far more suitable for determining the sequence of gaps and therefore ultimately finishing the sequence. As discussed above, the hierarchical approach has yielded a finished sequence, whereas Celera have not finished the sequencing, although Celera did subsequently publish a slightly improved assembly with “only” 210,000 gaps. 7.10 High-throughput Pyrosequencing Recently, a faster and more cost effective high-throughput sequencing protocol has been developed that is not based on the Sanger dideoxy chain ter-
198 <strong>Gene</strong> <strong>Cloning</strong> Tag sequence CGTGTCAGTCGGGAGTCTAGAA Primer 1 dTTP CGTGTCAGTCGGGAGTCTAGAA T + Sulfurylase Luciferase PPi ATP Light 2 dCTP CGTGTCAGTCGGGAGTCTAGAA TC dATP 3 CGTGTCAGTCGGGAGTCTAGAA TCA dGTP 4 CGTGTCAGTCGGGAGTCTAGAA TCAG dTTP 5 CGTGTCAGTCGGGAGTCTAGAA TCAG dCTP 6 CGTGTCAGTCGGGAGTCTAGAA TCAGCCC dATP 7 CGTGTCAGTCGGGAGTCTAGAA TCAGCCC 3 2 1 0 3 2 1 0 3 2 1 0 3 2 1 0 3 2 1 0 3 2 1 0 3 2 1 0 Flowgram T T C T C A T C A G T C A G T T C A G T C T C A G T C A mination protocol. The sequencing reaction was first described several years ago, but only recently has a method been developed to adapt it for high-throughput sequencing. We will discuss the basic sequencing procedure and then look at how it has been developed to allow rapid sequencing of whole genomes. Pyrosequencing One of the products of DNA synthesis, in addition to the DNA chain itself, is pyrophosphate which is released every time a nucleotide is added to a
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Gene Cloning
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Published by: Taylor & Francis Grou
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vi Contents 3.14 Designing PCR Prim
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viii Contents 8.3 Identifying Eukar
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1 Introduction 1.1 The Beginning of
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Introduction 3 which also conveys a
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Introduction 5 thought, or the brin
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2 Genome Organization Learning outc
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Genome Organization 9 and not quite
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Genome Organization 11 both of whic
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Genome Organization 13 relative com
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Genome Organization 15 Box 2.2 Inte
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Genome Organization 17 Table 2.2 Hu
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Genome Organization 19 close to oth
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Genome Organization 21 individual g
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slower rate than junk DNA. (To be m
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Genome Organization 25 kb 0 2 4 6 8
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Genome Organization 27 cloning pred
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Genome Organization 29 (a) 1 2 3 4
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Genome Organization 31 interphase a
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Genome Organization 33 A2.3. Chromo
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3 Key Tools for Gene Cloning Learni
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Key Tools for Gene Cloning 37 repli
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Key Tools for Gene Cloning 39 Box 3
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Key Tools for Gene Cloning 41 (a) (
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Key Tools for Gene Cloning 43 then,
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Key Tools for Gene Cloning 45 Figur
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Key Tools for Gene Cloning 47 Clear
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Key Tools for Gene Cloning 49 that
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Key Tools for Gene Cloning 51 same
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3.9 More About Vectors Detecting su
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Key Tools for Gene Cloning 55 in th
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Key Tools for Gene Cloning 57 Q3.13
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Key Tools for Gene Cloning 59 alway
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Key Tools for Gene Cloning 61 Figur
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Key Tools for Gene Cloning 63 (a) C
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Key Tools for Gene Cloning 65 Box 3
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Key Tools for Gene Cloning 67 simpl
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Key Tools for Gene Cloning 69 (a) 5
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Key Tools for Gene Cloning 71 92 92
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Key Tools for Gene Cloning 73 polym
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Key Tools for Gene Cloning 75 (a) T
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Key Tools for Gene Cloning 77 DNA s
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Key Tools for Gene Cloning 79 Q3.7.
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Key Tools for Gene Cloning 81 A3.15
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Key Tools for Gene Cloning 83 A3.20
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86 Gene Cloning you extract DNA fro
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88 Gene Cloning Box 4.1 Preparation
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90 Gene Cloning which is 6.3 Mb, to
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92 Gene Cloning Also, if all the fr
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94 Gene Cloning (a) (b) Bacterial s
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96 Gene Cloning (a) Insertion vecto
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98 Gene Cloning The strictly define
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100 Gene Cloning BamHI Ap r Tet r p
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102 Gene Cloning parB Cm r parA rep
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104 Gene Cloning (a) TTTTT AAAAA TT
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106 Gene Cloning Box 4.3 Converting
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108 Gene Cloning of your gene is. A
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110 Gene Cloning A4.2. Extract chro
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112 Gene Cloning A4.8. See Section
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114 Gene Cloning Q4.15. What are th
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116 Gene Cloning dystrophin gene is
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118 Gene Cloning it is still a form
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120 Gene Cloning One obvious proble
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122 Gene Cloning The main difficult
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124 Gene Cloning (a) Labeled single
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126 Gene Cloning N terminus Phe Val
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128 Gene Cloning Box 5.3 Types of D
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130 Gene Cloning Box 5.4 Methods fo
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132 Gene Cloning the DNA probe. Bot
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134 Gene Cloning a b c d e f g h 1
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136 Gene Cloning S. cerevisiae, and
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138 Gene Cloning Q5.7. Would you ex
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140 Gene Cloning use your genomic c
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142 Gene Cloning diseases, where th
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144 Gene Cloning (a) Replicative Ta
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Page 228 and 229: Bioinformatics 217 conjunction with
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Page 234 and 235: Bioinformatics 223 Matrix: EBLOSUM6
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Bioinformatics 247 EMBL Nucleotide
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250 Gene Cloning What sorts of stud
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252 Gene Cloning 9.2 Requirements f
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254 Gene Cloning (a) The lac promot
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256 Gene Cloning lac: CCAGGCTTTACAC
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258 Gene Cloning phage (such as T7)
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260 Gene Cloning 9.4 Some Problems
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262 Gene Cloning Figure 9.6 Inclusi
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264 Gene Cloning Box 9.1 Translatio
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266 Gene Cloning Box 9.2 Signal Seq
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268 Gene Cloning Ribosome Cytosol P
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270 Gene Cloning Other yeast specie
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272 Gene Cloning (turned on by an i
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274 Gene Cloning 9.6 A Final Word A
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276 Gene Cloning study, as they wil
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10 Gene Cloning in the Functional A
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Gene Cloning in the Functional Anal
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316 Gene Cloning genomic sequences
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318 Gene Cloning the first exon of
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320 Gene Cloning Box 11.1 End Label
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322 Gene Cloning mRNA 5' 3' 3' 5' S
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324 Gene Cloning mRNA 5' 3' 3' 5' G
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326 Gene Cloning cloning step. RACE
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328 Gene Cloning Prepare RNA from c
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330 Gene Cloning Gene A condition 1
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332 Gene Cloning (a) (b) (c) Immobi
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334 Gene Cloning tat and one in whi
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336 Gene Cloning Box 11.3 Examples
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338 Gene Cloning (a) MCS for promot
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340 Gene Cloning important regulato
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342 Gene Cloning (a) F DP DPA DNA-t
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344 Gene Cloning DNA alone DNA-prot
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346 Gene Cloning [FNR] GA - + -90 -
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348 Gene Cloning (a) 1 2 3 4 5 Prom
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350 Gene Cloning bind the transcrip
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352 Gene Cloning Target sequence Ye
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354 Gene Cloning have been develope
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356 Gene Cloning detected by autora
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358 Gene Cloning vide a complete li
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360 Gene Cloning MS 503 750 1400 16
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362 Gene Cloning Q11.3. Why is it e
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364 Gene Cloning (transcriptomics o
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366 Gene Cloning Figure 12.1 Transg
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368 Gene Cloning Herbicide Non-tran
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370 Gene Cloning that has been used
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372 Gene Cloning tumors with high f
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374 Gene Cloning Transgene encoding
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376 Gene Cloning placed on the mark
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378 Gene Cloning Gene A: a gene whi
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380 Gene Cloning Isolate the gene o
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382 Gene Cloning Box 12.1 Recombina
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384 Gene Cloning damage to the DNA
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386 Gene Cloning Box 12.2 The Produ
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388 Gene Cloning as to whether the
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390 Gene Cloning transformed. The i
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392 Gene Cloning 1. Gene of interes
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394 Gene Cloning Showing that the D
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396 Gene Cloning complete plants by
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398 Gene Cloning 12.5 Knockout Mice
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400 Gene Cloning introduced back in
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402 Gene Cloning Chromosome Constru
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404 Gene Cloning cells, and one has
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406 Gene Cloning mapped by inverse
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408 Gene Cloning traditional method
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410 Gene Cloning Further Reading Th
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412 Gene Cloning (a) Maternal chrom
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414 Gene Cloning chromosomes togeth
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416 Gene Cloning Box 13.1 DNA Profi
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418 Gene Cloning Although forensic
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420 Gene Cloning Dye terminators (a
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422 Gene Cloning With the advent of
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424 Gene Cloning Box 13.2 Genetic A
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426 Gene Cloning Box 13.3 Methods o
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428 Gene Cloning (a) PCR primer seq
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430 Gene Cloning (a) Target specifi
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432 Gene Cloning (a) R Q 5' 3' (b)
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434 Gene Cloning liquid chromatogra
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436 Gene Cloning such as pre-implan
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438 Gene Cloning attack there is a
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440 Gene Cloning and also because l
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442 Gene Cloning John Mary Ann Pete
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444 Gene Cloning monitoring the pro
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446 Gene Cloning Codon Three nucleo
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448 Gene Cloning Homologous recombi
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450 Gene Cloning Phenotype The obse
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452 Gene Cloning Vector A self-repl
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454 Gene Cloning Capillary electrop
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456 Gene Cloning EST see expressed
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458 Gene Cloning Metallothionein, 3
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460 Gene Cloning Protein addition o
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462 Gene Cloning Transcription, ide
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Gene Cloning

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