Gene Cloning

More documents

Recommendations

Info

Bioinformatics 227 (a) DB:ID Source Length Identity% Ungapped% Overlap E SW:CTR2_CANFA Chymotrypsinogen 2 precursor (EC 263 86.692 86.692 263 1.8e-103 SW:CTRB1_HUMAN Chymotrypsinogen B precursor (EC 263 85.932 85.932 263 1.33-101 SW:CTRB1_RAT Chymotrypsinogen B precursor (EC 3 263 84.411 84.411 263 2.8e-101 SW:CTRB_BOVIN Chymotrypsinogen B (EC 3.4.21.1). 245 81.224 81.224 245 2.4e-91 SW:CTRA_BOVIN Chymotrypsinogen A (EC 3.4.21.1). 245 76.735 76.735 245 1.3e-84 SW:CTRA_GADMO Chymotrypsin A precursor (EC 3.4. 263 67.755 67.755 245 2.0e-76 SW:CTRB_GADMO Chymotrypsin B (EC 3.4.21.1). 245 67.073 67.623 246 1.6e-74 SW:CTRL_HUMAN Chymotrypsin-like protease CTRL-1 264 55.682 55.894 264 9.0e-68 SW:ELA2_PIG Elastase 2 precursor (EC 3.4.21.71) 269 44.444 47.619 270 1.0e-42 SW:ELA2_RAT Elastase 2 precursor (EC 3.4.21.71) 271 43.333 46.063 270 8.2e-42 SW:CLCR_HUMAN Caldecrin precursor (EC 3.4.21.2) 268 42.471 44.898 259 1.1e-41 SW:ELA2A_HUMAN Elastase 2A precursor (EC 3.4.21 269 43.911 47.410 271 1.9e-40 SW:ELA2_MOUSE Elastase 2 precursor (EC 3.4.21.7 271 41.481 44.094 270 5.9e-40 SW:CLCR_RAT Caldecrin precursor (EC 3.4.21.2) 268 41.699 44.082 259 1.1e-39 SW:ELA2_BOVIN Elastase 2 precursor (EC 3.4.21.7 269 43.333 46.429 270 3.9e-39 SW:TMPS9_MOUSE Transmembrane protease, serine 9 1065 40.784 42.623 255 8.5e-38 SW:ELA2B_HUMAN Elastase 2B precursor (EC 3.4.21 269 41.544 45.200 272 1.7e-37 SW:TMPS9_RAT Transmembrane protease, serine 9 1061 40.000 41.803 255 5.6e-37 SW:TMPS3_MOUSE Transmembrane protease, serine 3 453 40.711 44.017 253 7.7e-37 SW:PLMN_CANFA Plasminogen (EC 3.4.21.7)(Fragme 333 38.314 41.667 261 1.2e-36 (b) >>SW:CTR2_CANFA PO4813 Chymotrypsinogen 2 precursor (EC (263 aa) initn: 1607 initl: 1607 opt: 1607 Z-score: 1984.4 bits: 374.8 E(): 1.8e-103 Smith-Waterman score: 1607; 86.692% identity (86.692% ungapped) in 263 aa overlap (1-263:1-263) 10 20 30 40 50 60 Query MAFLWLLSCFALVGAASSCGVPAIEPVLNGLSRIVNGEDAVPGSWPWQVSLQDSTGFHFC ::::::::::::.:.: .::::::.:::.::::::::::::::::::::::::::::::: SW:CTR MAFLWLLSCFALLGTAFGCGVPAIQPVLSGLSRIVNGEDAVPGSWPWQVSLQDSTGFHFC 10 20 30 40 50 60 70 80 90 100 110 120 Query GGSLISEDWVVTAAHCGVRTSHLVVAGEFDQSSSEENIQVLKIAEVFKNPKFNMFTVRND ::::::::::::::::::::.: ::::::::.:. :.:::::::.:::::::::::. :: SW:CTR GGSLISEDWVVTAAHCGVRTTHQVVAGEFDQGSDAESIQVLKIAKVFKNPKFNMFTINND 70 80 90 100 110 120 130 140 150 160 170 180 Query ITLLKLATPARFSETVSPVCLPQATDEFPPGLMCVTTGWGRTKYNANKTPDKLQQAALPL :::::::::::::.::: ::::::::.:: : .::::::: ::.. .:::::::::::: SW:CTR ITLLKLATPARFSKTVSAVCLPQATDDFPAGTLCVTTGWGLTKHTNANTPDKLQQAALPL 130 140 150 160 170 180 190 200 210 220 230 240 Query LSNAECKKFWGSKITDVMICAGASGVSSCMGDSGGPLVCQKDGAWTLVGIVSWGSGTCST ::::::::::::::::.:.::::::::::::::::::::::::::::::::::::::::: SW:CTR LSNAECKKFWGSKITDLMVCAGASGVSSCMGDSGGPLVCQKDGAWTLVGIVSWGSGTCST 190 200 210 220 230 240 250 260 Query SVPAVYSRVTELIPWVQEILAAN :.:.::.:::.::::::.:: :: SW:CTR STPGVYARVTKLIPWVQQILQAN 250 260 Figure 8.12 Excerpts from a Fasta output showing a search with a canine chymotrypsinogen amino acid sequence against the SwissProt database. a) The summary table lists, in order, the sequences from the SwissProt database which most closely match the query sequence. This excerpt shows the top 20 matches although the full output would normally list 50 or 100. b) The first pair-wise alignment showing the similarity between the query sequence and the most similar sequence in the database. Exact matches between the two sequences are indicated by :, conservative changes, as defined by a blosum 50 matrix by • .
228 <strong>Gene</strong> <strong>Cloning</strong> the query sequence, and that the region of similarity between the two sequences is over most of the sequence. Between 87% and 38% of the amino acids are identical in each pair of sequences. While the percentage of identical amino acids gives you some idea of how significant the matches are, the E value in the final column is the most important piece of statistical data. The “expect” or E value for a match between two sequences is a measure of how likely it is that the match has occurred by chance when searching a database of a particular size. In this example there is a 1.8 × 10 –103 chance that the first match could have occurred by chance, in other words the alignment between these two sequences is statistically highly significant. There are some useful rules of thumb which can help in evaluating E values although because these are statistical measures they are only a tool and as always need to be used in conjunction with knowledge of the biology of the proteins involved. In general, an E value of 0.02 is considered to be significant although, as in the example in Figure 8.12, E values are often very much smaller. An E value of 1 indicates that, when searching a database of this size, you would expect one match, this good, just by chance; it is therefore very unlikely to have any biological or evolutionary significance. E values of between 0.02 and 1 fall in what is known as the twilight zone, within which you will need your knowledge of the biology of the genes and proteins involved in order to assess the significance of the match. It is in the twilight zone that exciting discoveries showing relationships between proteins previously thought to be unrelated are being made. In addition to the summary table, Fasta also displays pair-wise alignments between the query sequence and each of the sequences that match it (Figure 8.12b). Examination of this comparison shows that the query sequence and the match with the smallest E value are very similar along their entire lengths with no gaps in the alignment and very few substitutions, most of these being with amino acids with similar properties. 8.11 BLAST This program performs much the same task as Fasta although the computational approach is different. The BLAST output also gives a summary of the sequences which most closely match the query sequence (Figure 8.13a). Again, E values indicate how significant the matches are and hyperlinks point to other databases. Some implementations of BLAST, such as at NCBI (the US National Center for Biotechnology Information) also provide a graphical overview (Figure 8.13c) which can be particularly useful when, as in the example shown, some matches are only to part of the sequence. Another useful feature, which is often combined with BLAST, is the ability to look for the occurrence of conserved domains in the sequence (Figure 8.13d). Conserved domains will be discussed in more detail in Section 8.15.
Page 2:
Gene Cloning
Page 5 and 6:
Published by: Taylor & Francis Grou
Page 7 and 8:
vi Contents 3.14 Designing PCR Prim
Page 9 and 10:
viii Contents 8.3 Identifying Eukar
Page 12 and 13:
1 Introduction 1.1 The Beginning of
Page 14 and 15:
Introduction 3 which also conveys a
Page 16 and 17:
Introduction 5 thought, or the brin
Page 18 and 19:
2 Genome Organization Learning outc
Page 20 and 21:
Genome Organization 9 and not quite
Page 22 and 23:
Genome Organization 11 both of whic
Page 24 and 25:
Genome Organization 13 relative com
Page 26 and 27:
Genome Organization 15 Box 2.2 Inte
Page 28 and 29:
Genome Organization 17 Table 2.2 Hu
Page 30 and 31:
Genome Organization 19 close to oth
Page 32 and 33:
Genome Organization 21 individual g
Page 34 and 35:
slower rate than junk DNA. (To be m
Page 36 and 37:
Genome Organization 25 kb 0 2 4 6 8
Page 38 and 39:
Genome Organization 27 cloning pred
Page 40 and 41:
Genome Organization 29 (a) 1 2 3 4
Page 42 and 43:
Genome Organization 31 interphase a
Page 44 and 45:
Genome Organization 33 A2.3. Chromo
Page 46 and 47:
3 Key Tools for Gene Cloning Learni
Page 48 and 49:
Key Tools for Gene Cloning 37 repli
Page 50 and 51:
Key Tools for Gene Cloning 39 Box 3
Page 52 and 53:
Key Tools for Gene Cloning 41 (a) (
Page 54 and 55:
Key Tools for Gene Cloning 43 then,
Page 56 and 57:
Key Tools for Gene Cloning 45 Figur
Page 58 and 59:
Key Tools for Gene Cloning 47 Clear
Page 60 and 61:
Key Tools for Gene Cloning 49 that
Page 62 and 63:
Key Tools for Gene Cloning 51 same
Page 64 and 65:
3.9 More About Vectors Detecting su
Page 66 and 67:
Key Tools for Gene Cloning 55 in th
Page 68 and 69:
Key Tools for Gene Cloning 57 Q3.13
Page 70 and 71:
Key Tools for Gene Cloning 59 alway
Page 72 and 73:
Key Tools for Gene Cloning 61 Figur
Page 74 and 75:
Key Tools for Gene Cloning 63 (a) C
Page 76 and 77:
Key Tools for Gene Cloning 65 Box 3
Page 78 and 79:
Key Tools for Gene Cloning 67 simpl
Page 80 and 81:
Key Tools for Gene Cloning 69 (a) 5
Page 82 and 83:
Key Tools for Gene Cloning 71 92 92
Page 84 and 85:
Key Tools for Gene Cloning 73 polym
Page 86 and 87:
Key Tools for Gene Cloning 75 (a) T
Page 88 and 89:
Key Tools for Gene Cloning 77 DNA s
Page 90 and 91:
Key Tools for Gene Cloning 79 Q3.7.
Page 92 and 93:
Key Tools for Gene Cloning 81 A3.15
Page 94:
Key Tools for Gene Cloning 83 A3.20
Page 97 and 98:
86 Gene Cloning you extract DNA fro
Page 99 and 100:
88 Gene Cloning Box 4.1 Preparation
Page 101 and 102:
90 Gene Cloning which is 6.3 Mb, to
Page 103 and 104:
92 Gene Cloning Also, if all the fr
Page 105 and 106:
94 Gene Cloning (a) (b) Bacterial s
Page 107 and 108:
96 Gene Cloning (a) Insertion vecto
Page 109 and 110:
98 Gene Cloning The strictly define
Page 111 and 112:
100 Gene Cloning BamHI Ap r Tet r p
Page 113 and 114:
102 Gene Cloning parB Cm r parA rep
Page 115 and 116:
104 Gene Cloning (a) TTTTT AAAAA TT
Page 117 and 118:
106 Gene Cloning Box 4.3 Converting
Page 119 and 120:
108 Gene Cloning of your gene is. A
Page 121 and 122:
110 Gene Cloning A4.2. Extract chro
Page 123 and 124:
112 Gene Cloning A4.8. See Section
Page 125 and 126:
114 Gene Cloning Q4.15. What are th
Page 127 and 128:
116 Gene Cloning dystrophin gene is
Page 129 and 130:
118 Gene Cloning it is still a form
Page 131 and 132:
120 Gene Cloning One obvious proble
Page 133 and 134:
122 Gene Cloning The main difficult
Page 135 and 136:
124 Gene Cloning (a) Labeled single
Page 137 and 138:
126 Gene Cloning N terminus Phe Val
Page 139 and 140:
128 Gene Cloning Box 5.3 Types of D
Page 141 and 142:
130 Gene Cloning Box 5.4 Methods fo
Page 143 and 144:
132 Gene Cloning the DNA probe. Bot
Page 145 and 146:
134 Gene Cloning a b c d e f g h 1
Page 147 and 148:
136 Gene Cloning S. cerevisiae, and
Page 149 and 150:
138 Gene Cloning Q5.7. Would you ex
Page 151 and 152:
140 Gene Cloning use your genomic c
Page 153 and 154:
142 Gene Cloning diseases, where th
Page 155 and 156:
144 Gene Cloning (a) Replicative Ta
Page 157 and 158:
146 Gene Cloning where the transpos
Page 159 and 160:
148 Gene Cloning DNA fragment in th
Page 161 and 162:
150 Gene Cloning Bacteria transform
Page 163 and 164:
152 Gene Cloning Wild-type gene Mut
Page 165 and 166:
154 Gene Cloning molecule using PCR
Page 167 and 168:
156 Gene Cloning digesting chromoso
Page 169 and 170:
158 Gene Cloning + + Ac Avr Avr cf-
Page 171 and 172:
160 Gene Cloning which are very clo
Page 173 and 174:
162 Gene Cloning Box 6.3 Restrictio
Page 175 and 176:
164 Gene Cloning Initial region clo
Page 177 and 178:
166 Gene Cloning Box 6.4 Nonsense S
Page 179 and 180:
168 Gene Cloning 6.10 Cloning of th
Page 181 and 182:
170 Gene Cloning are two possible o
Page 183 and 184:
172 Gene Cloning Isolation of the t
Page 185 and 186:
174 Gene Cloning Two approaches wer
Page 187 and 188: 176 Gene Cloning synthesize DNA de
Page 189 and 190: 178 Gene Cloning Box 7.1 Denaturing
Page 191 and 192: 180 Gene Cloning G A T C Figure 7.4
Page 193 and 194: 182 Gene Cloning although other con
Page 195 and 196: 184 Gene Cloning A large DNA fragme
Page 197 and 198: 186 Gene Cloning chain reaction, fl
Page 199 and 200: 188 Gene Cloning rounds of amplific
Page 201 and 202: 190 Gene Cloning Contig 2 Clone 12
Page 203 and 204: 192 Gene Cloning λ Clone Scaffold
Page 205 and 206: 194 Gene Cloning Genomic DNA (a) Cl
Page 207 and 208: 196 Gene Cloning therefore sequence
Page 209 and 210: 198 Gene Cloning Tag sequence CGTGT
Page 211 and 212: 200 Gene Cloning Genomic DNA (a) Ad
Page 213 and 214: 202 Gene Cloning bases. The light s
Page 215 and 216: 204 Gene Cloning A7.3. (a) The dide
Page 218 and 219: 8 Bioinformatics Learning outcomes:
Page 220 and 221: Bioinformatics 209 Table 8.1 The ge
Page 222 and 223: Bioinformatics 211 5' accgcgcatggtg
Page 224 and 225: Bioinformatics 213 Correlation Scor
Page 226 and 227: Bioinformatics 215 AGG T R A G T C
Page 228 and 229: Bioinformatics 217 conjunction with
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
Page 240 and 241: Bioinformatics 229 (a) Sequences pr
Page 242 and 243: Bioinformatics 231 list is also of
Page 244 and 245: Bioinformatics 233 this relates to
Page 246 and 247: Bioinformatics 235 common functiona
Page 248 and 249: Bioinformatics 237 These two regula
Page 250 and 251: Bioinformatics 239 three-dimensiona
Page 252 and 253: Bioinformatics 241 Neisseria mening
Page 254 and 255: Bioinformatics 243 A8.1. The triple
Page 256 and 257: Bioinformatics 245 Q8.11. Which gro
Page 258: Bioinformatics 247 EMBL Nucleotide
Page 261 and 262: 250 Gene Cloning What sorts of stud
Page 263 and 264: 252 Gene Cloning 9.2 Requirements f
Page 265 and 266: 254 Gene Cloning (a) The lac promot
Page 267 and 268: 256 Gene Cloning lac: CCAGGCTTTACAC
Page 269 and 270: 258 Gene Cloning phage (such as T7)
Page 271 and 272: 260 Gene Cloning 9.4 Some Problems
Page 273 and 274: 262 Gene Cloning Figure 9.6 Inclusi
Page 275 and 276: 264 Gene Cloning Box 9.1 Translatio
Page 277 and 278: 266 Gene Cloning Box 9.2 Signal Seq
Page 279 and 280: 268 Gene Cloning Ribosome Cytosol P
Page 281 and 282: 270 Gene Cloning Other yeast specie
Page 283 and 284: 272 Gene Cloning (turned on by an i
Page 285 and 286: 274 Gene Cloning 9.6 A Final Word A
Page 287 and 288: 276 Gene Cloning study, as they wil
Page 290 and 291:
10 Gene Cloning in the Functional A
Page 292 and 293:
Gene Cloning in the Functional Anal
Page 294 and 295:
Page 296 and 297:
Page 298 and 299:
Page 300 and 301:
Page 302 and 303:
Page 304 and 305:
Page 306 and 307:
Page 308 and 309:
Page 310 and 311:
Page 312 and 313:
Page 314 and 315:
Page 316 and 317:
Page 318 and 319:
Page 320 and 321:
Page 322 and 323:
Page 324:
Page 327 and 328:
316 Gene Cloning genomic sequences
Page 329 and 330:
318 Gene Cloning the first exon of
Page 331 and 332:
320 Gene Cloning Box 11.1 End Label
Page 333 and 334:
322 Gene Cloning mRNA 5' 3' 3' 5' S
Page 335 and 336:
324 Gene Cloning mRNA 5' 3' 3' 5' G
Page 337 and 338:
326 Gene Cloning cloning step. RACE
Page 339 and 340:
328 Gene Cloning Prepare RNA from c
Page 341 and 342:
330 Gene Cloning Gene A condition 1
Page 343 and 344:
332 Gene Cloning (a) (b) (c) Immobi
Page 345 and 346:
334 Gene Cloning tat and one in whi
Page 347 and 348:
336 Gene Cloning Box 11.3 Examples
Page 349 and 350:
338 Gene Cloning (a) MCS for promot
Page 351 and 352:
340 Gene Cloning important regulato
Page 353 and 354:
342 Gene Cloning (a) F DP DPA DNA-t
Page 355 and 356:
344 Gene Cloning DNA alone DNA-prot
Page 357 and 358:
346 Gene Cloning [FNR] GA - + -90 -
Page 359 and 360:
348 Gene Cloning (a) 1 2 3 4 5 Prom
Page 361 and 362:
350 Gene Cloning bind the transcrip
Page 363 and 364:
352 Gene Cloning Target sequence Ye
Page 365 and 366:
354 Gene Cloning have been develope
Page 367 and 368:
356 Gene Cloning detected by autora
Page 369 and 370:
358 Gene Cloning vide a complete li
Page 371 and 372:
360 Gene Cloning MS 503 750 1400 16
Page 373 and 374:
362 Gene Cloning Q11.3. Why is it e
Page 375 and 376:
364 Gene Cloning (transcriptomics o
Page 377 and 378:
366 Gene Cloning Figure 12.1 Transg
Page 379 and 380:
368 Gene Cloning Herbicide Non-tran
Page 381 and 382:
370 Gene Cloning that has been used
Page 383 and 384:
372 Gene Cloning tumors with high f
Page 385 and 386:
374 Gene Cloning Transgene encoding
Page 387 and 388:
376 Gene Cloning placed on the mark
Page 389 and 390:
378 Gene Cloning Gene A: a gene whi
Page 391 and 392:
380 Gene Cloning Isolate the gene o
Page 393 and 394:
382 Gene Cloning Box 12.1 Recombina
Page 395 and 396:
384 Gene Cloning damage to the DNA
Page 397 and 398:
386 Gene Cloning Box 12.2 The Produ
Page 399 and 400:
388 Gene Cloning as to whether the
Page 401 and 402:
390 Gene Cloning transformed. The i
Page 403 and 404:
392 Gene Cloning 1. Gene of interes
Page 405 and 406:
394 Gene Cloning Showing that the D
Page 407 and 408:
396 Gene Cloning complete plants by
Page 409 and 410:
398 Gene Cloning 12.5 Knockout Mice
Page 411 and 412:
400 Gene Cloning introduced back in
Page 413 and 414:
402 Gene Cloning Chromosome Constru
Page 415 and 416:
404 Gene Cloning cells, and one has
Page 417 and 418:
406 Gene Cloning mapped by inverse
Page 419 and 420:
408 Gene Cloning traditional method
Page 421 and 422:
410 Gene Cloning Further Reading Th
Page 423 and 424:
412 Gene Cloning (a) Maternal chrom
Page 425 and 426:
414 Gene Cloning chromosomes togeth
Page 427 and 428:
416 Gene Cloning Box 13.1 DNA Profi
Page 429 and 430:
418 Gene Cloning Although forensic
Page 431 and 432:
420 Gene Cloning Dye terminators (a
Page 433 and 434:
422 Gene Cloning With the advent of
Page 435 and 436:
424 Gene Cloning Box 13.2 Genetic A
Page 437 and 438:
426 Gene Cloning Box 13.3 Methods o
Page 439 and 440:
428 Gene Cloning (a) PCR primer seq
Page 441 and 442:
430 Gene Cloning (a) Target specifi
Page 443 and 444:
432 Gene Cloning (a) R Q 5' 3' (b)
Page 445 and 446:
434 Gene Cloning liquid chromatogra
Page 447 and 448:
436 Gene Cloning such as pre-implan
Page 449 and 450:
438 Gene Cloning attack there is a
Page 451 and 452:
440 Gene Cloning and also because l
Page 453 and 454:
442 Gene Cloning John Mary Ann Pete
Page 455 and 456:
444 Gene Cloning monitoring the pro
Page 457 and 458:
446 Gene Cloning Codon Three nucleo
Page 459 and 460:
448 Gene Cloning Homologous recombi
Page 461 and 462:
450 Gene Cloning Phenotype The obse
Page 463 and 464:
452 Gene Cloning Vector A self-repl
Page 465 and 466:
454 Gene Cloning Capillary electrop
Page 467 and 468:
456 Gene Cloning EST see expressed
Page 469 and 470:
458 Gene Cloning Metallothionein, 3
Page 471 and 472:
460 Gene Cloning Protein addition o
Page 473:
462 Gene Cloning Transcription, ide
show all

Gene Cloning

Create successful ePaper yourself

Delete template?

Save as template?