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Gene Cloning and DNA Analysis 5/e
[번역서: 유전자클로닝과 DNA 분석 -입문서]
판매가격  : 25,000원
적립금  : 750점
출판사  : Blackwell Science
저자  : Brown
발행일  : 2006년
페이지 수  : 408면
ISBN  : 1405111216
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Chapter 1 Why Gene Cloning and DNA Analysis are Important

1.1 The early development of genetics

1.2 The advent of gene cloning and the polymerase chain reaction

1.3 What is gene cloning?

1.4 What is PCR?

1.5 Why gene cloning and PCR are so important

1.5.1 Gene isolation by cloning

1.5.2 Gene isolation by PCR

1.6 How to find your way through this book

Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages

2.1 Plasmids

2.1.1 Basic features of plasmids

2.1.2 Size and copy number

2.1.3 Conjugation and compatibility

2.1.4 Plasmid classification

2.1.5 Plasmids in organisms other than bacteria

2.2 Bacteriophages

2.2.1 Basic features of bacteriophages

2.2.2 Lysogenic phages

Gene organization in l DNA molecule

The linear and circular forms of l DNA

M13 - a filamentous phage

The attraction of M13 as a cloning vector

2.2.3 Viruses as cloning vectors for other organisms

Chapter 3 Purification of DNA from Living Cells

3.1 Preparation of total cell DNA

3.1.1 Growing and harvesting a bacterial culture

3.1.2 Preparation of a cell extract

3.1.3 Purification of DNA from a cell extract

Removing contaminants by organic extraction and enzyme digestion

Using ion-exchange chromatography to purify DNA from a cell extract

3.1.4 Concentration of DNA samples

3.1.5 Measurement of DNA concentration

3.1.6 Other methods for the preparation of total cell DNA

3.2 Preparation of plasmid DNA

3.2.1 Separation on the basis of size

3.2.2 Separation on the basis of conformation

Alkaline denaturation

Ethidium bromide-caesium chloride density gradient centrifugation

3.2.3 Plasmid amplification

3.3 Preparation of bacteriophage DNA

3.3.1 Growth of cultures to obtain a high l titre

3.3.2 Preparation of non-lysogenic l phages

3.3.3 Collection of phages from an infected culture

3.3.4 Purification of DNA from l phage particles

3.3.5 Purification of M13 DNA causes few problems

Chapter 4 Manipulation of Purified DNA

4.1 The range of DNA manipulative enzymes

4.1.1 Nucleases

4.1.2 Ligases

4.1.3 Polymerases

4.1.4 DNA modifying enzymes

4.1.5 Topoisomerases

4.2 Enzymes for cutting DNA - restriction endonucleases

4.2.1 The discovery and function of restriction endonucleases

4.2.2 Type II restriction endonucleases cut DNA at specific nucleotide sequences

4.2.3 Blunt ends and sticky ends

4.2.4 The frequency of recognition sequences in a DNA molecule

4.2.5 Performing a restriction digest in the laboratory

4.2.6 Analysing the result of restriction endonuclease cleavage

Separation of molecules by gel electrophoresis

Visualizing DNA molecules by staining a gel

Visualizing DNA molecules by autoradiography

4.2.7 Estimation of the sizes of DNA molecules

4.2.8 Mapping the positions of different restriction sites in a DNA molecule

4.3 Ligation - joining DNA molecules together

4.3.1 The mode of action of DNA ligase

4.3.2 Sticky ends increase the efficiency of ligation

4.3.3 Putting sticky ends onto a blunt-ended molecule



Chapter 5 Introduction of DNA into Living Cells

5.1 Transformation - the uptake of DNA by bacterial cells

5.1.1 Not all species of bacteria are equally efficient at DNA uptake

5.1.2 Preparation of competent E. coli cells

5.1.3 Selection for transformed cells

5.2 Identification of recombinants

5.2.1 Recombinant selection with pBR322 - insertional inactivation of an antibiotic

resistance gene

5.2.2 Insertional inactivation does not always involve antibiotic resistance

5.3 Introduction of phage DNA into bacterial cells

5.3.1 Transfection

5.3.2 In vitro packaging of l cloning vectors

5.3.3 Phage infection is visualized as plaques on an agar medium

5.4 Identification of recombinant phages

5.4.1 Insertional inactivation of a lacZ´ gene carried by the phage vector

5.4.2 Insertional inactivation of the l cI gene

5.4.3 Selection using the Spi phenotype

5.4.4 Selection on the basis of l genome size

5.5 Introduction of DNA into non-bacterial cells

5.5.1 Transformation of individual cells

5.5.2 Transformation of whole organisms

Producing sticky ends by homopolymer tailing

Chapter 6 Cloning Vectors for E. coli

6.1 Cloning vectors based on E. coli plasmids

6.1.1 The nomenclature of plasmid cloning vectors

6.1.2 The useful properties of pBR322

6.1.3 The pedigree of pBR322

6.1.4 More sophisticated E. coli plasmid cloning vectors

pUC8 - a Lac selection plasmid

pGEM3Z - in vitro transcription of cloned DNA

6.2 Cloning vectors based on M13 bacteriophage

6.2.1 Development of the M13 cloning vectors

M13mp7 - symmetrical cloning sites

More complex M13 vectors

6.2.2 Hybrid plasmid-M13 vectors

6.3 Cloning vectors based on l bacteriophage

6.3.1 Segments of the l genome can be deleted without impairing viability

6.3.2 Natural selection can be used to isolate modified l that lack certain restriction


6.3.3 Insertion and replacement vectors

Insertion vectors

Replacement vectors

6.3.4 Cloning experiments with l insertion or replacement vectors

6.3.5 Long DNA fragments can be cloned using a cosmid

6.4 l and other high capacity vectors enable genomic libraries to be constructed

6.5 Vectors for other bacteria

Chapter 7 Cloning Vectors for Eukaryotes

7.1 Vectors for yeast and other fungi

7.1.1 Selectable markers for the 2µm plasmid

7.1.2 Vectors based on the 2 µm plasmid - yeast episomal plasmids

7.1.3 A YEp may insert into yeast chromosomal DNA

7.1.4 Other types of yeast cloning vector

7.1.5 Artificial chromosomes can be used to clone long pieces of DNA in yeast

The structure and use of a YAC vector

Applications for YAC vectors

7.1.6 Vectors for other yeasts and fungi

7.2 Cloning vectors for higher plants

7.2.1 Agrobacterium tumefaciens - nature's smallest genetic engineer

Using the Ti plasmid to introduce new genes into a plant cell

Production of transformed plants with the Ti plasmid

The Ri plasmid

Limitations of cloning with Agrobacterium plasmids

7.2.2 Cloning genes in plants by direct gene transfer

Direct gene transfer into the nucleus

Transfer of genes into the chloroplast genome

7.2.3 Attempts to use plant viruses as cloning vectors

Caulimovirus vectors

Geminivirus vectors

7.3 Cloning vectors for animals

7.3.1 Cloning vectors for insects

P elements as cloning vectors for Drosophila

Cloning vectors based on insect viruses

7.3.2 Cloning in mammals

Cloning vectors for mammals

Gene cloning without a vector

Chapter 8 How to Obtain a Clone of a Specific Gene

8.1 The problem of selection

8.1.1 There are two basic strategies for obtaining the clone you want

8.2 Direct selection

8.2.1 Marker rescue extends the scope of direct selection

8.2.2 The scope and limitations of marker rescue

8.3 Identification of a clone from a gene library

8.3.1 Gene libraries

8.3.2 Not all genes are expressed at the same time

8.3.3 mRNA can be cloned as complementary DNA

8.4 Methods for clone identification

8.4.1 Complementary nucleic acid strands hybridize to each other

8.4.2 Colony and plaque hybridization probing

8.4.3 Examples of the practical use of hybridization probing

Abundancy probing to analyse a cDNA library

Oligonucleotide probes for genes whose translation products have been


Heterologous probing allows related genes to be identified

8.4.4 Identification methods based on detection of the translation product of the

cloned gene

Antibodies are required for immunological detection methods

Using a purified antibody to detect protein in recombinant colonies

The problem of gene expression

Chapter 9 The Polymerase Chain Reaction

9.1 The polymerase chain reaction in outline

9.2 PCR in more detail

9.2.1 Designing the oligonucleotide primers for a PCR

9.2.2 Working out the correct temperatures to use

9.2.3 After the PCR: studying PCR products

Gel electrophoresis of PCR products

Cloning PCR products

9.3 Problems with the error rate of Taq polymerase


Chapter 10 Studying Gene Location and Structure

10.1 How to study the location of a gene

10.1.1 Locating the position of a gene on a small DNA molecule

10.1.2 Locating the position of a gene on a large DNA molecule

Separating chromosomes by gel electrophoresis

In situ hybridization to visualize the position of a gene on a eukaryotic


10.2 DNA sequencing - working out the structure of a gene

10.2.1 The Sanger-Coulson method - chain-terminating nucleotides

The primer

Synthesis of the complementary strand

Four separate reactions result in four families of terminated strands

Reading the DNA sequence from the autoradiograph

Not all DNA polymerases can be used for sequencing

10.2.2 Automated DNA sequencing

10.2.3 Sequencing PCR products

10.2.4 The Maxam-Gilbert method - chemical degradation of DNA

10.2.5 Building up a long DNA sequence

10.2.6 The achievements of DNA sequencing

Chapter 11 Studying Gene Expression and Function

11.1 Studying the transcript of a cloned gene

11.1.1 Electron microscopy of nucleic acid molecules

11.1.2 Analysis of DNA-RNA hybrids by nuclease treatment

11.1.3 Transcript analysis by primer extension

11.1.4 Other techniques for studying RNA transcripts

Northern hybridization

Reverse transcription-PCR (RT-PCR)

Rapid amplification of cDNA ends (RACE)

RNA sequencing

11.2 Studying the regulation of gene expression

11.2.1 Identifying protein binding sites on a DNA molecule

Gel retardation of DNA-protein complexes

Footprinting with DNase I

Modification interference assays

11.2.2 Identifying control sequences by deletion analysis

Reporter genes

Carrying out a deletion analysis

11.3 Identifying and studying the translation product of a cloned gene

11.3.1 HRT and HART can identify the translation product of a cloned gene

11.3.2 Analysis of proteins by in vitro mutagenesis

Different types of in vitro mutagenesis techniques

Using an oligonucleotide to create a point mutation in a cloned gene

Other methods of creating a point mutation in a cloned gene

The potential of in vitro mutagenesis

11.3.3 Studying protein-protein interactions

Phage display

The yeast two hybrid system

Chapter 12 Studying Genomes

12.1 Genomics - how to sequence a genome

12.1.1 The shotgun approach to genome sequencing

The H. influenzae genome sequencing project

Problems with shotgun cloning

12.1.2 The clone contig approach

Clone contig assembly by chromosome walking

Rapid methods for clone contig assembly

Clone contig assembly by sequence tagged site content analysis

12.1.3 Using a map to aid sequence assembly

Genetic maps

Physical maps

The importance of a map in sequence assembly

12.2 Post-genomics - trying to understand a genome sequence

12.2.1 Identifying the genes in a genome sequence

Searching for open reading frames

Distinguishing real genes from chance ORFs

12.2.2 Determining the function of an unknown gene

12.3 Studies of the transcriptome and proteome

12.3.1 Studying the transcriptome

12.3.2 Studying the proteome


Chapter 13 Production of Protein from Cloned Genes

13.1 Special vectors for expression of foreign genes in E. coli

13.1.1 The promoter is the critical component of an expression vector

The promoter must be chosen with care

Examples of promoters used in expression vectors

13.1.2 Cassettes and gene fusions

13.2 General problems with the production of recombinant protein in E. coli

13.2.1 Problems resulting from the sequence of the foreign gene

13.2.2 Problems caused by E. coli

13.3 Production of recombinant protein by eukaryotic cells

13.3.1 Recombinant protein from yeast and filamentous fungi

Saccharomyces cerevisiae as the host for recombinant protein synthesis

Other yeasts and fungi

13.3.2 Using animal cells for recombinant protein production

Protein production in mammalian cells

Protein production in insect cells

13.3.3 Pharming - recombinant protein from live animals and plants

Pharming in animals

Recombinant proteins from plants

Ethical concerns raised by pharming

Chapter 14 Gene Cloning and DNA Analysis in Medicine

14.1 Production of recombinant pharmaceuticals

14.1.1 Recombinant insulin

Synthesis and expression of artificial insulin genes

14.1.2 Synthesis of human growth hormones in E. coli

14.1.3 Recombinant factor VIII

14.1.4 Synthesis of other recombinant human proteins

14.1.5 Recombinant vaccines

Producing vaccines as recombinant proteins

Recombinant vaccines in transgenic plants

Live recombinant virus vaccines

14.2 Identification of genes responsible for human diseases

14.2.1 How to identify a gene for a genetic disease

Locating the approximate position of the gene in the human genome

Identification of candidates for the disease gene

14.3 Gene therapy

14.3.1 Gene therapy for inherited diseases

14.3.2 Gene therapy and cancer

14.3.3 The ethical issues raised by gene therapy

Chapter 15 Gene Cloning and DNA Analysis in Agriculture

15.1 The gene addition approach to plant genetic engineering

15.1.1 Plants that make their own insecticides

The d-endotoxins of Bacillus thuringiensis

Cloning a d-endotoxin gene in maize

Cloning d-endotoxin genes in chloroplasts

Countering insect resistance to d-endotoxin crops

15.1.2 Herbicide resistant crops

'Roundup Ready' crops

A new generation of glyphosate resistant crops

15.1.2 Other gene addition projects

15.2 Gene subtraction

15.2.1 The principle behind antisense technology

15.2.2 Antisense RNA and the engineering of fruit ripening in tomato

Using antisense RNA to inactivate the polygalacturonase gene

Using antisense RNA to inactivate ethylene synthesis

15.2.3 Other examples of the use of antisense RNA in plant genetic engineering

15.3 Problems with genetically modified plants

15.3.1 Safety concerns with selectable markers

15.3.2 The terminator technology

15.3.3 The possibility of harmful effects on the environment

Chapter 16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology

16.1 DNA analysis in the identification of crime suspects

16.1.1 Genetic fingerprinting by hybridization probing

16.1.2 DNA profiling by PCR of short tandem repeats

16.2 Studying kinship by DNA profiling

16.2.1 Related individuals have similar DNA profiles

16.2.2 DNA profiling and the remains of the Romanovs

STR analysis of the Romanov bones

The missing children

16.3 Sex identification by DNA analysis

16.3.1 PCRs directed at Y chromosome-specific sequences

16.3.2 PCR of the amelogenin gene

16.4 Archaeogenetics - using DNA to study human evolution

16.4.1 The origins of modern humans

DNA analysis has challenged the multiregional hypothesis

DNA analysis shows that Neanderthals are not the ancestors of modern


16.4.2 DNA can also be used to study prehistoric human migrations

The spread of agriculture into Europe

Using mitochondrial DNA to study past human migrations into Europe

{교재 사용시 강의 자료 문의 바랍니다.}
제품명 Gene Cloning and DNA Analysis 5/e
판매가격 25,000원
제조사 Blackwell Science
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