Gene Cloning

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Bioinformatics 229 (a) Sequences producing significant alignments: (bits) Value gi|130316|sp|P00747|PLMN_HUMAN Plasminogen precursor [Conta... 1652 0.0 gi|114062|sp|P08519|APOA_HUMAN Apolipoprotein(a) precursor ... 1035 0.0 gi|123114|sp|P26927|HGFL_HUMAN Hepatocyte growth factor-lik... 472 e-133 gi|123116|sp|P14210|HGF_HUMAN Hepatocyte growth factor prec... 459 e-129 gi|137119|sp|P00750|TPA_HUMAN Tissue-type plasminogen activ... 236 2e-62 gi|123057|sp|P05981|HEPS_HUMAN Serine protease hepsin (Tran... 197 2e-50 gi|13124582|sp|P57727|TMPS3_HUMAN Transmembrane protease, s... 192 3e-49 gi|18203319|sp|Q9NRR2|TRYG1_HUMAN Tryptase gamma precursor ... 191 7e-49 gi|18202734|sp|Q9BQR3|PRS27_HUMAN Pancreasin precursor (Mar... 191 9e-49 gi|547643|sp|Q04756|HGFA_HUMAN Hepatocyte growth factor act... 191 9e-49 ------------- gi|9296990|sp|Q9UKR3|KLK13 HUMAN Kallikrein 13 precursor (K... 143 3e-34 gi|19860140|sp|P48740|MASP1_HUMAN Complement-activating com... 137 2e-32 gi|3915626|sp|P00746|CFAD_HUMAN Complement factor D precurs... 133 2e-31 gi|7387859|sp|O00187|MASP2_HUMAN Mannan-binding lectin seri... 129 4e-30 gi|3914477|sp|O43240|KLK10_HUMAN Kallikrein 10 precursor (P... 106 4e-23 gi|20978519|sp|Q96MU8|KREM1_HUMAN Kremen protein 1 precurso... 69 5e-12 gi|6226950|sp|Q99767|APBA2_HUMAN Amyloid beta A4 precursor ... 38 0.012 gi|38258267|sp|Q9UK17|KCD3_HUMAN Potassium voltage-gated ch... 33 0.40 gi|45477126|sp|Q86X10|K1219_HUMAN Protein KIAA1219 30 3.4 gi|2497674|sp|Q16527|CSR2_HUMAN Cysteine and glycine-rich p... 29 5.8 (b) >gi|130316|sp|P00747|PLMN_HUMAN Plasminogen precursor [Contains: Angiostatin] Length = 810 Score = 1652 bits (4278), Expect = 0.0 Identities = 760/810 (93%), Positives = 760/810 (93%) Query: 1 MEHKEVVXXXXXXXKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIXXXXXXXXXXXXFT 60 MEHKEVV KSGQGEPLDDYVNTQGASLFSVTKKQLGAGSI FT Sbjct: 1 MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFT 60 Query: 61 CRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKN 120 CRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTNSKTKN Sbjct: 61 CRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKN 120 Query: 121 GITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILE 180 GITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILE Sbjct: 121 GITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILE 180 (c) Distribution of 92 Blast Hits on the Query Sequence Mouse-over to show defline and scores. Click to show alignments Color Key for Alignment Scores =200 1_26469 0 100 200 300 400 500 600 700 800
230 <strong>Gene</strong> <strong>Cloning</strong> (d) 1 100 200 300 400 500 600 700 810 KR KR KR KR KR Tryp_SPc Figure 8.13 Excerpts from a BLAST output showing a search with the blood clotting protein precursor plasminogen against the human sequences in the SwissProt database. a) The summary table lists the best matches between the query sequence and the human sequences in SwissProt. BLAST would normally list up to 100 matches, the first and matches 41–50 are shown here. b) Part of the first pair-wise alignment showing the similarity between the query sequence and the most similar sequence in the database. Sequences of low complexity are excluded from the analysis and are marked by X in the query sequence. c) Graphical overview showing the alignments between the database sequences and the query. d) Graphical representation showing putative conserved domains detected in the query sequence. Most scientists have a preference for using either Fasta or BLAST; some may have good reasons for this preference and for some it may just be habit. In practice, for the student, both programs will do a good job and there is probably little to choose between them. However, it is good practice to use both programs, particularly if you are looking at matches in the twilight zone. Because both programs use methods designed to speed up what would otherwise be a very computationally intensive process, they may miss matches in this region. Using both programs is one way to minimize the risk of missing important matches. 8.12 What Can Similarity Searches Tell Us About the Biology of the Sequences Being Compared? As with pair-wise alignments, when we find a match between our query sequence and other sequences we can infer that the proteins are likely to have a similar structure and consequently a similar function. We also infer an evolutionary relationship: proteins with similar sequences are likely to be homologues, and to have evolved from a common ancestor. Their evolutionary relationship is evident from the similarity observed in their sequences and reinforced if it can be demonstrated that they have similar structures and functions. In the case of a sequence about which nothing is known, for instance a sequence generated during one of the genome sequencing projects, a similarity search, in conjunction with techniques which look for the characteristics of genes, will help to identify the protein. The query sequence used in the comparison shown in Figure 8.12 is that of canine chymotrypsinogen, the precursor of an important digestive enzyme chymotrypsin. In this case the sequence is taken from a canine genome sequencing project. You can see that the sequence at the top of the
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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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166 Gene Cloning Box 6.4 Nonsense S
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168 Gene Cloning 6.10 Cloning of th
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170 Gene Cloning are two possible o
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172 Gene Cloning Isolation of the t
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174 Gene Cloning Two approaches wer
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176 Gene Cloning synthesize DNA de
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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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