Gene Cloning

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Production of Proteins from Cloned Genes 253 Strong promoter Multiple cloning site Transcription terminator Ribosome binding site Origin of replication Selectable marker (e.g. antibiotic resistance) Figure 9.1 The key features of a typical vector for protein production. are discussed in detail in the next section. Several other features important in these plasmids, which are often referred to as expression vectors, are summarized in Figure 9.1 and discussed in more detail below. Promoters for expression Protein production requires the use of a promoter which can be used to drive expression of the gene to high levels. This promoter will be upstream of the gene of interest in a suitable plasmid vector, such that it directs transcription of the gene which encodes the protein of interest. A number of different promoter systems are widely used in E. coli, with the choice for any particular experiment depending on a variety of factors. The two most important features of a promoter are its strength and the degree to which expression from it can be regulated. The first is important in achieving high levels of expression: in general, the stronger the promoter, the more mRNA will be synthesized, and the more protein will result from the translation of this mRNA. The ability to control expression is also important, however, particularly as it is often the case that high levels of expression of a heterologous protein can lead to a decrease in growth of E. coli, or even death. Thus the ideal promoter for most expression experiments is one which is strong, but easy to turn down or off. The simplest examples of promoters for protein expression are those which are derived from operons of E. coli, with the lac promoter of the lac operon, and the ara promoter from the arabinose operon (also sometimes referred to as the pBAD promoter), being widely used (Figure 9.2). You may already know that expression from the lac promoter is regulated by the lac repressor protein, LacI (see Chapter 11 for more information on regulation of gene expression). In the absence of lactose LacI is bound to the DNA,
254 Gene Cloning (a) The lac promoter (i) Glucose absent, IPTG present: promoter ON Multiple mRNAs synthesized by RNA polymerase CRP protein RNA polymerase Gene of interest (ii) Glucose present, IPTG absent: promoter WEAKLY ON lac repressor Low transcription Gene of interest (b) The ara (or pBAD) promoter (i) Glucose absent, arabinose present: promoter ON AraC CRP protein AraC RNA polymerase Multiple mRNAs synthesized by RNA polymerase Gene of interest (ii) Glucose present, arabinose absent: promoter OFF AraC AraC No transcription Gene of interest Figure 9.2 The lac and ara promoters. The diagram illustrates their modes of action when on and off. CRP protein: a positive activator that binds in the absence of glucose. and hence prevents expression of the genes of the lac operon in the absence of lactose, which encode proteins for lactose uptake and cleavage. The lac promoter DNA which contains the binding sites for RNA polymerase can be cloned upstream of any gene, thus placing the expression of those genes under the control of the signals which normally direct lac gene
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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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178 Gene Cloning Box 7.1 Denaturing
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180 Gene Cloning G A T C Figure 7.4
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182 Gene Cloning although other con
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184 Gene Cloning A large DNA fragme
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186 Gene Cloning chain reaction, fl
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188 Gene Cloning rounds of amplific
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190 Gene Cloning Contig 2 Clone 12
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192 Gene Cloning λ Clone Scaffold
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194 Gene Cloning Genomic DNA (a) Cl
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196 Gene Cloning therefore sequence
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198 Gene Cloning Tag sequence CGTGT
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200 Gene Cloning Genomic DNA (a) Ad
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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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