Gene Cloning

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8 Bioinformatics Learning outcomes: By the end of this chapter you will have an understanding of: • how a DNA sequence can be searched for regions which could code for genes (open reading frames) and how these can be evaluated in both prokaryotic and eukaryotic organisms • how to interpret a pair-wise alignment between related sequences, showing an understanding of similarity and identity as applied to amino acids • the types of information stored in primary sequence databases, secondary pattern databases and structure databases • the two main programs used for similarity searching and how to interpret the output from them • how to interpret a multiple sequence alignment including identifying conserved regions • how to interpret regular expressions describing consensus patterns • what the three-dimensional structure of proteins can add to the understanding of the protein • how phylogenetic trees depict the relationships between organisms or species 8.1 Introduction Determining DNA sequences has become a routine procedure; specialist laboratories can sequence vast stretches of DNA each day and new wholegenome sequences are published regularly. Why are biologists so keen to know the sequence of the genes they study? Determining DNA sequence is not an end in itself but a milestone along the path to understanding a particular biological phenomenon. Unless you can interpret the DNA sequence, work out which parts of it are genes and what sorts of proteins these encode, then the information is a meaningless series of Gs, As, Ts and Cs. There are basically two approaches that you can take to interpreting a DNA sequence. Firstly, you can look at the piece of DNA sequence that you have determined and use what is known about the characteristics of a
208 Gene Cloning typical gene to work out from first principles which parts of the sequence are likely to code for proteins. The second and very powerful way of looking at DNA sequence uses homology searching, comparing the sequence with other DNA sequences, which are stored in vast public databases. Using a combination of these approaches can unlock an immense amount of information from a DNA sequence. Both of these approaches require the use of computers to help in the analysis of immense amounts of data, and together form the basis of the discipline which is called bioinformatics. Bioinformatics is a theoretical discipline in that it allows you to make predictions about DNA and protein sequence, but these need to be tested in the laboratory. Remember the flow of genetic information in a cell is from DNA to RNA and finally to proteins. The DNA is transcribed into discrete, essentially gene-sized, pieces of RNA which are then translated into a sequence of amino acids. These will fold into a complex three-dimensional protein structure and may be further modified before they are fully matured into functioning proteins. However, despite the fact that proteins are not translated directly from DNA, most sequence analysis is performed on the DNA sequence and so we will adopt the conventions of DNA rather than RNA in the discussions in this chapter, referring to thymine rather than uracil. Q8.1. What is the DNA triplet codon for methionine? What would the codon be in an RNA molecule? Refer to Table 8.1, which shows the genetic code. Q8.2. What do the codons UAA, UAG and UGA encode? 8.2 What Does a Gene Look Like? The DNA sequence of a gene is the sequences which encode proteins together with the sequences which regulate gene expression (Figure 8.1). In bioinformatics terms any region of DNA sequence without a stop codon could potentially encode a protein; these regions are called open reading frames (ORFs). If you examine the sequence in Figure 8.2, you should be able to see that even in this very short sequence stop codons occur in frames 1 and 3. The sequence in frame 3, however, could encode part of a gene. Most computer algorithms designed to look for ORFs use the simple principle of looking for regions without stop codons. Q8.3. Why are three alternative translations given below the sequence in Figure 8.2? Q8.4. Are the three alternative translations given in Figure 8.2 the only possible translations of this sequence?
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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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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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10 Gene Cloning in the Functional A
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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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336 Gene Cloning Box 11.3 Examples
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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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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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372 Gene Cloning tumors with high f
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374 Gene Cloning Transgene encoding
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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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392 Gene Cloning 1. Gene of interes
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394 Gene Cloning Showing that the D
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398 Gene Cloning 12.5 Knockout Mice
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402 Gene Cloning Chromosome Constru
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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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422 Gene Cloning With the advent of
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424 Gene Cloning Box 13.2 Genetic A
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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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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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