Gene Cloning and DNA Analysis: An Introduction, 5th Edition

by Terry A. Brown (University of Manchester)

• ISBN13: 9781405111218
• ISBN10: 1405111216
• Edition: 5th
• Format: Paperback
• Copyright: 2006-03-01
• Publisher: Wiley-Blackwell
• More Book Details
• Purchase Benefits

Summary

The fourth edition of Gene Cloning is a fully revised and updated version of this most popular introductory text on the subject. This exciting new edition retains the basic structure and organisation that has proved successful for the previous three editions.Two major changes in the contents of the book are the introduction of the subject of the polymerase chain reaction (PCR) at the beginning of the book with more emphasis being placed on PCR throughout and the inclusion of a new chapter on genome sequencing and functional analysis.

Author Biography

Terry Brown, University of Manchester, Faculty of Life Sciences, UK

Table of Contents

Preface to the Fifth Edition	xv
Preface to the Fourth Edition	xvi
Preface to the Third Edition	xvii
Preface to the Second Edition	xviii
Preface to the First Edition	xx
Part 1: The Basic Principles of Gene Cloning and DNA Analysis	1	(196)
Why Gene Cloning and DNA Analysis are Important	3	(11)
The early development of genetics	3	(1)
The advent of gene cloning and the polymerase chain reaction	4	(1)
What is gene cloning?	5	(1)
What is PCR?	6	(2)
Why gene cloning and PCR are so important	8	(4)
Gene isolation by cloning	8	(1)
Gene isolation by PCR	9	(3)
How to find your way through this book	12	(2)
Vectors for Gene Cloning: Plasmids and Bacteriophages	14	(14)
Plasmids	14	(5)
Basic features of plasmids	14	(2)
Size and copy number	16	(1)
Conjugation and compatibility	17	(1)
Plasmid classification	18	(1)
Plasmids in organisms other than bacteria	19	(1)
Bacteriophages	19	(9)
Basic features of bacteriophages	19	(1)
Lysogenic phages	20	(2)
Gene organization in the λ DNA molecule	22	(1)
The linear and circular forms of λ DNA	23	(2)
M13 -- a filamentous phage	25	(1)
The attraction of M13 as a cloning vector	25	(2)
Viruses as cloning vectors for other organisms	27	(1)
Purification of DNA from Living Cells	28	(26)
Preparation of total cell DNA	28	(11)
Growing and harvesting a bacterial culture	29	(1)
Preparation, of a cell extract	30	(3)
Purification of DNA from a cell extract	33	(1)
Removing contaminants by organic extraction and - enzyme digestion	34	(1)
Using ion-exchange chromatography to purify DNA from a cell extract	35	(1)
Concentration of DNA samples	35	(2)
Measurement of DNA concentration	37	(1)
Other methods for the preparation of total cell DNA	37	(2)
Preparation of plasmid DNA	39	(8)
Separation on the basis of size	41	(1)
Separation on the basis of conformation	42	(1)
Alkaline denaturation	43	(1)
Ethidium bromide-caesium chloride density gradient centrifugation	43	(3)
Plasmid amplification	46	(1)
Preparation of bacteriophage DNA	47	(7)
Growth of cultures to obtain a high λ titre	47	(1)
Preparation of non-lysogenic λ phages	48	(2)
Collection of phages from an infected culture	50	(1)
Purification of DNA from λ phage particles	51	(1)
Purification of M13 DNA causes few problems	52	(2)
Manipulation of Purified DNA	54	(33)
The range of DNA manipulative enzymes	55	(6)
Nucleases	55	(2)
Ligases	57	(1)
Polymerases	58	(2)
DNA modifying enzymes	60	(1)
Topoisomerases	61	(1)
Enzymes for cutting DNA -- restriction endonucleases	61	(17)
The discovery and function of restriction -- endonucleases	63	(1)
Type II restriction endonucleases cut DNA at specific nucleotide sequences	64	(1)
Blunt ends and sticky ends	64	(1)
The frequency of recognition sequences in a DNA molecule	65	(3)
Performing a restriction digest in the laboratory	68	(2)
Analysing the result of restriction endonuclease cleavage	70	(1)
Separation of molecules by gel electrophoresis	70	(1)
Visualizing DNA molecules by staining a gel	70	(2)
Visualizing DNA molecules by autoradiography	72	(2)
Estimation of the sizes of DNA molecules	74	(2)
Mapping the positions of different restriction sites in a DNA molecule	76	(2)
Ligation -- joining DNA molecules together	78	(9)
The mode of action of DNA ligase	78	(1)
Sticky ends increase the efficiency of ligation	78	(2)
Putting sticky ends onto a blunt-ended molecule	80	(1)
Linkers	80	(1)
Adaptors	80	(4)
Producing sticky ends by homopolymer tailing	84	(3)
Introduction of DNA into Living Cells	87	(20)
Transformation -- the uptake of DNA by bacterial cells	90	(3)
Not all species of bacteria are equally efficient at DNA uptake	90	(1)
Preparation of competent E. coli cells	90	(1)
Selection for transformed cells	91	(2)
Identification of recombinants	93	(4)
Recombinant selection with pBR322 -- insertional inactivation of an antibiotic resistance gene	93	(2)
Insertional inactivation does not always involve antibiotic resistance	95	(2)
Introduction of phage DNA into bacterial cells	97	(4)
Transfection	98	(1)
In vitro packaging of λ cloning vectors	98	(1)
Phage infection is visualized as plaques on an agar medium	99	(2)
Identification of recombinant phages	101	(2)
Insertional inactivation of a lacZ' gene carried by the phage vector	101	(1)
Insertional inactivation of the λ cl gene	101	(2)
Selection using the Spi phenotype	103	(1)
Selection on the basis of λ genome size	103	(1)
Introduction of DNA into non-bacterial cells	103	(4)
Transformation of individual cells	103	(3)
Transformation of whole organisms	106	(1)
Cloning Vectors for E. coli	107	(25)
Cloning vectors based on E. coli plasmids	108	(6)
The nomenclature of plasmid cloning vectors	108	(1)
The useful properties of pBR322	108	(1)
The pedigree of pBR322	109	(1)
More sophisticated E. coli plasmid cloning vectors	109	(2)
pUC8 -- a Lac selection plasmid	111	(2)
pGEM3Z -- in vitro transcription of cloned DNA	113	(1)
Cloning vectors based on M13 bacteriophage	114	(6)
Development of the M13 cloning vectors	115	(1)
M13mp7 -- symmetrical cloning sites	115	(3)
More complex M13 vectors	118	(2)
Hybrid plasmid-M13 vectors	120	(1)
Cloning vectors based on λ bacteriophage	120	(9)
Segments of the λ genome can be deleted without impairing viability	122	(1)
Natural selection can be used to isolate modified λ that lack certain restriction sites	123	(1)
Insertion and replacement vectors	123	(1)
Insertion vectors	123	(1)
Replacement vectors	124	(2)
Cloning experiments with λ insertion or replacement vectors	126	(1)
Long DNA fragments can be cloned using a cosmid	127	(2)
λ and other high capacity vectors enable genomic libraries, to be constructed	129	(1)
Vectors for other bacteria	130	(2)
Cloning Vectors for Eukaryotes	132	(26)
Vectors for yeast and other fungi	132	(9)
Selectable markers for the 2 um plasmid	132	(1)
Vectors based on the 2 um plasmid -- yeast episomal plasmids	133	(2)
A YEp may insert into yeast chromosomal DNA	135	(1)
Other types of yeast cloning vector	135	(3)
Artificial chromosomes can be used to clone long pieces of DNA in yeast	138	(1)
The structure and use of a YAC vector	138	(2)
Applications for YAC vectors	140	(1)
Vectors for other yeasts and fungi	140	(1)
Cloning vectors for higher plants	141	(10)
Agrobacterium tumefaciens -- nature's smallest genetic engineer	141	(1)
Using the Ti plasmid to introduce new genes into a plant cell	142	(3)
Production of transformed plants with the Ti plasmid	145	(1)
The Ri plasmid	145	(1)
Limitations of cloning with Agrobacterium plasmids	146	(1)
Cloning genes in plants by direct gene transfer	147	(1)
Direct gene transfer into the nucleus	147	(2)
Transfer of genes into the chloroplast genome	149	(1)
Attempts to use plant viruses as cloning vectors	150	(1)
Caulimovirus vectors	150	(1)
Geminivirus vectors	150	(1)
Cloning vectors for animals	151	(7)
Cloning vectors for insects	151	(1)
Pelements as cloning vectors for Drosophila	151	(2)
Cloning vectors based on insect viruses	153	(1)
Cloning in mammals	153	(1)
Cloning vectors for mammals	153	(2)
Gene cloning without a vector	155	(3)
How to Obtain a Clone of a Specific Gene	158	(23)
The problem of selection	158	(1)
There are two basic strategies for obtaining the clone you want	158	(1)
Direct selection	159	(4)
Marker rescue extends the scope of direct selection	161	(2)
The scope and limitations of marker rescue	163	(1)
Identification of a clone from a gene library	163	(3)
Gene libraries	163	(1)
Not all genes are expressed at the same time	164	(2)
mRNA can be cloned as complementary DNA	166	(1)
Methods for clone identification	166	(15)
Complementary nucleic acid strands hybridize to each other	166	(2)
Colony and plaque hybridization probing	168	(4)
Examples of the practical use of hybridization probing	172	(1)
Abundancy probing to analyse a cDNA library	172	(1)
Oligonucleotide probes for genes whose translation products have been characterised	172	(4)
Heterologous probing allows related genes to be identified	176	(1)
Identification methods based on detection of the - translation product of the cloned gene	177	(1)
Antibodies are required for immunological detection methods	177	(1)
Using a purified antibody to detect protein in recombinant colonies	178	(1)
The problem of gene expression	178	(3)
The Polymerase Chain Reaction	181	(16)
The polymerase chain reaction in outline	181	(3)
PCR in more detail	184	(9)
Designing the oligonucleotide primers for a PCR	184	(2)
Working out the correct temperatures to use	186	(3)
After the PCR: studying PCR products	189	(1)
Gel electrophoresis of PCR products	189	(1)
Cloning PCR products	190	(3)
Problems with the error rate of Taq polymerase	193	(4)
Part 2: The Applications of Gene Cloning and DNA Analysis in Research	197	(78)
Studying Gene Location and Structure	199	(21)
How to study the location of a gene	199	(8)
Locating the position of a gene on a small DNA molecule	199	(3)
Locating the position of a gene on a large DNA molecule	202	(1)
Separating chromosomes by gel electrophoresis	203	(2)
In situ hybridization to visualize the position of a gene on a eukaryotic chromosome	205	(2)
DNA sequencing -- working out the structure of a gene	207	(13)
The Sanger--Coulson method -- chain-terminating nucleotides	207	(1)
The primer	207	(1)
Synthesis of the complementary strand	207	(2)
Four separate reactions result in four families of terminated strands	209	(1)
Reading the DNA sequence from the autoradiograph	209	(1)
Not all DNA polymerases can be used for sequencing	210	(1)
Automated DNA sequencing	211	(2)
Sequencing PCR products	213	(1)
The Maxam--Gilbert method -- chemical degradation of DNA	214	(2)
Building up a long DNA sequence	216	(2)
The achievements of DNA sequencing	218	(2)
Studying Gene Expression and Function	220	(32)
Studying the transcript of a cloned gene	221	(9)
Electron microscopy of nucleic acid molecules	222	(1)
Analysis of DNA-RNA hybrids by nuclease treatment	223	(1)
Transcript analysis by primer, extension	224	(1)
Other techniques for studying RNA transcripts	225	(1)
Northern hybridization	225	(1)
Reverse transcription--PCR (RT-PCR)	226	(1)
Rapid amplification of cDNA ends (RACE)	227	(1)
RNA sequencing	228	(2)
Studying the regulation of gene expression	230	(9)
Identifying protein binding sites on a DNA molecule	231	(1)
Gel retardation of DNA--protein complexes	231	(1)
Footprinting with DNase I	232	(1)
Modification interference assays	233	(3)
Identifying control sequences by deletion analysis	236	(1)
Reporter genes	236	(1)
Carrying out a deletion analysis	237	(2)
Identifying and studying the translation product of a cloned gene	239	(13)
HRT and HART can identify the translation product of a cloned gene	239	(1)
Analysis of proteins by in vitro mutagenesis	240	(1)
Different types of in vitro mutagenesis techniques	241	(2)
Using an oligonucleotide to create a point mutation in a cloned gene	243	(2)
Other methods of creating a point mutation in a cloned gene	245	(2)
The potential of in vitro mutagenesis	247	(1)
Studying protein--protein interactions	247	(1)
Phage display	247	(3)
The yeast two hybrid system	250	(2)
Studying Genomes	252	(23)
Genomics -- how to sequence a genome	253	(11)
The shotgun approach to genome sequencing	254	(1)
The H. influenzae genome sequencing project	255	(2)
Problems with shotgun cloning	257	(1)
The clone contig approach	257	(1)
Clone contig assembly by chromosome walking	258	(1)
Rapid methods for clone-contig assembly	259	(1)
Clone contig assembly by sequence tagged site content analysis	260	(1)
Using a map to aid sequence assembly	261	(1)
Genetic maps	261	(1)
Physical maps	262	(2)
The importance of a map in sequence assembly	264	(1)
Post-genomics -- trying to understand a genome sequence	264	(5)
Identifying the genes in a genome sequence	265	(1)
Searching for open reading frames	265	(1)
Distinguishing real genes from chance ORFs	266	(2)
Determining the function of an unknown gene	268	(1)
Studies of the transcriptome and proteome	269	(6)
Studying the transcriptome	269	(2)
Studying the proteome	271	(4)
Part 3: The Applications of Gene Cloning and DNA Analysis in Biotechnology	275	(88)
Production of Protein from Cloned Genes	277	(25)
Special vectors for expression of foreign genes in E. coli	279	(8)
The promoter is the critical component of an expression vector	282	(1)
The promoter must be chosen with care	282	(2)
Examples of promoters used in expression vectors	284	(1)
Cassettes and gene fusions	285	(2)
General problems with the production of recombinant protein in E. coli	287	(5)
Problems resulting from the sequence of the foreign gene	288	(3)
Problems caused by E. coli	291	(1)
Production of recombinant protein by eukaryotic cells	292	(10)
Recombinant protein from yeast and filamentous fungi	292	(1)
Saccharomyces cerevisiae as the host for recombinant protein synthesis	292	(1)
Other yeasts and fungi	293	(2)
Using animal cells for recombinant protein production	295	(1)
Protein production in mammalian cells	295	(1)
Protein production in insect, cells	295	(1)
Pharming -- recombinant protein from live animals and plants	296	(1)
Pharming in animals	297	(1)
Recombinant proteins from plants	298	(1)
Ethical concerns raised by pharming	299	(3)
Gene Cloning and DNA Analysis in Medicine	302	(21)
Production of recombinant pharmaceuticals	302	(12)
Recombinant insulin	302	(1)
Synthesis and expression of artificial insulin genes	303	(2)
Synthesis of human growth hormones in E. coli	305	(1)
Recombinant factor VIII	306	(3)
Synthesis of other recombinant human proteins	309	(1)
Recombinant vaccines	309	(1)
Producing vaccines as recombinant proteins	310	(2)
Recombinant vaccines in transgenic plants	312	(1)
Live recombinant virus vaccines	312	(2)
Identification of genes responsible for human diseases	314	(5)
How to identify a gene for a genetic disease	315	(1)
Locating the approximate position of the gene in the human genome	315	(3)
Identification of candidates for the disease gene	318	(1)
Gene therapy	319	(4)
Gene therapy for inherited diseases	319	(2)
Gene therapy and cancer	321	(1)
The ethical issues raised by gene therapy	321	(2)
Gene Cloning and DNA Analysis in Agriculture	323	(23)
The gene addition approach to plant genetic engineering	324	(10)
Plants that make their own insecticides	324	(1)
The δ-endotoxins of Bacillus thuringiensis	324	(1)
Cloning a δ--endotoxin gene in maize	325	(3)
Cloning δ-endotoxin genes in chloroplasts	328	(1)
Countering insect resistance to δ-endotoxin crops	329	(2)
Herbicide resistant crops	331	(1)
`Roundup Ready' crops	331	(1)
A new generation of glyphosate resistant crops	332	(2)
Other gene addition projects	334	(1)
Gene subtraction	334	(7)
The principle behind antisense technology	335	(1)
Antisense RNA and the engineering of fruit ripening in tomato	336	(1)
Using antisense RNA to inactivate the polygalacturonase gene	337	(2)
Using antisense RNA to inactivate ethylene synthesis	339	(1)
Other examples of the use of antisense RNA in plant genetic engineering	340	(1)
Problems with genetically modified plants	341	(5)
Safety concerns with selectable markers	341	(1)
The terminator technology	342	(2)
The possibility of harmful effects on the environment	344	(2)
Gene Cloning and DNA Analysis in Forensic Science and Archaeology	346	(17)
DNA analysis in the identification of crime suspects	346	(4)
Genetic fingerprinting by hybridization probing	347	(1)
DNA profiling by PCR of short tandem repeats	347	(3)
Studying kinship by DNA profiling	350	(3)
Related individuals have similar DNA profiles	350	(1)
DNA profiling and the remains of the Romanovs	350	(1)
STR analysis of the Romanov bones	351	(1)
The missing children	352	(1)
Sex identification by DNA analysis	353	(3)
PCRs directed at Y chromosome-specific sequences	353	(1)
PCR of the amelogenin gene	354	(2)
Archaeogenetics -- using DNA to study human evolution	356	(7)
The origins of modern humans	356	(1)
DNA analysis has challenged the multiregional hypothesis	357	(1)
DNA analysis shows that Neanderthals are not the ancestors of modern Europeans	358	(1)
DNA can also be used to study prehistoric human migrations	359	(1)
The spread of agriculture into Europe	359	(1)
Using mitochondrial DNA to study past human migrations into Europe	360	(3)
Glossary	363	(16)
Index	379