Gene Cloning

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Bioinformatics 217 conjunction with everything else that you know about the genes you are looking for. In the final analysis this type of bioinformatics analysis can only help you to formulate a theory which can then be tested in the laboratory. This type of sequence analysis can only take us so far in interpreting the sequence data. The next step in the analysis is to compare our sequence with that of other known sequences. This can give us information about the evolutionary relationship between sequences, as well as about their structure and function. 8.4 Sequence Comparisons Sequence comparison is a very powerful tool; its use is based on the assumption that proteins, which have evolved from a common ancestor, will have similar sequences. This is a phenomenon called homology. If you can show that your sequence belongs to a group of similar sequences, then you can deduce that it has evolved from the same ancestral sequence; it is also likely that it has a similar function to the other members of the group. 8.5 Pair-wise Comparisons A pair-wise comparison basically involves lining up two sequences with each other and observing how similar they are. Have a look at the two amino acid sequences in Figure 8.6a; you can probably see some similarities between the two sequences. You should be able to spot three runs of at least three amino acids that are identical in the two sequences. The sequences are not exactly the same length and the spacing between the regions that match up vary so it is necessary to introduce two gaps to make an optimal alignment of the sequences Figure 8.6b. With short sequences like these it is reasonably easy to align them by eye, with longer sequences you need to use an objective and automatable method to do this. The first step in this process is to prepare a grid like that in Figure 8.7. Each filled-in square in the grid represents a position where the two sequences are identical. The regions where the two sequences are identical show up as diagonal lines. Because there are only 20 amino acids you would expect to see some positions where any two randomly chosen sequences were identical. These matches are not relevant in an evolutionary sense and introduce noise into (a) SVKLQDRKLQDRMKSGKVSVKLQD VSVKLQDQLSQRLQDNSKMVSVKLQD (b) SVKLQD---RKLQDRMKSGKVSVKLQD |||||| ||| | ||||||| VSVKLQDQLSQRLQDNSK--MVSVKLQD Figure 8.6 Pair-wise alignment of amino acid sequences. a) Shows two arbitrary amino acid sequences. b) The best alignment of the two sequences, vertical bars represent identical amino acids.
218 <strong>Gene</strong> <strong>Cloning</strong> (a) V S V K L Q D Q L S Q R L Q D N S K M V S V K L Q D S V K L Q D R K L Q D R M K S G K V S V K L Q D Figure 8.7 Grids for pair-wise sequence comparison. a) The first sequence is written along the top of the grid and the second sequence down the left hand side. Wherever the two sequences are identical the corresponding square on the grid is filled in. (b) V S V K L Q D Q L S Q R L Q D N S K M V S V K L Q D S V K L Q D R K L Q D R M K S G K V S V K L Q D Figure 8.7. b) The grid shows positions where there are at least three consecutive amino acids which are identical in both sequences.
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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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146 Gene Cloning where the transpos
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148 Gene Cloning DNA fragment in th
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150 Gene Cloning Bacteria transform
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152 Gene Cloning Wild-type gene Mut
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154 Gene Cloning molecule using PCR
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156 Gene Cloning digesting chromoso
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158 Gene Cloning + + Ac Avr Avr cf-
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160 Gene Cloning which are very clo
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162 Gene Cloning Box 6.3 Restrictio
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164 Gene Cloning Initial region clo
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Page 218 and 219: 8 Bioinformatics Learning outcomes:
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Page 230 and 231: Bioinformatics 219 (c) V S V K L Q
Page 232 and 233: Bioinformatics 221 Tiny P Small Ali
Page 234 and 235: Bioinformatics 223 Matrix: EBLOSUM6
Page 236 and 237: Bioinformatics 225 Box 8.2 DNA and
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Page 240 and 241: Bioinformatics 229 (a) Sequences pr
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Page 254 and 255: Bioinformatics 243 A8.1. The triple
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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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