We are living in an era of genomics and biotechnology revolution. To many molecular and cellular biologists, the flood of information on human genomes and new methodologies is particularly overwhelming because biotechnology is forging ahead and bringing about rapid changes continuously. Because every organism depends on molecular action for survival, molecular biology research has become more dominant in multiple disciplines. In fact, it is a general tendency that, when the National Institutes of Health and other funding agencies award grants, they give high priority to research proposals that employ molecular biology approaches. How is it possible to catch up, receive updates on new biotechnology, and use the most recent, proven techniques for novel research? One of the aims of this book is to provide investigators with the tools needed for modern molecular and cellular biology research.

Another goal is to guide graduate students in their thesis research. In our experience, good graduate training mandates independent performance with minimum advice from a mentor. How is a novel research project for a thesis selected? What are the hypotheses, objectives and experimental designs? How can technical problems be grasped and current techniques mastered? Where does one begin and what are the predicted results? A graduate student needs some help with these questions; this book will provide the clues.

This book covers a wide range of current biotechnology methods developed and widely used in molecular biology, biochemistry, cell biology and immunology. The methods and protocols described in the appropriate chapters include:

• Strategies for novel research projects

• Rapid isolation of specific cDNAs or genes by PCR

• Construction and screening of cDNA and genomic DNA libraries

• Preparation of DNA constructs

• Nonisotopic and isotopic DNA or RNA sequencing

• Information superhighway and computer databases of nucleic acids and proteins

• Characterization of DNA, RNA or proteins by Southern, northern or western blot hybridization

• Gene overexpression, gene underexpression and gene knockout in mammalian systems

• Analysis of DNA or abundance of mRNA by radioactivity in situ hybridization (RISH)

• Localization of DNA or abundance mRNA by fluorescence in situ hybridization (FISH)

• In situ PCR hybridization of low copy genes and in situ RT-PCR detection of low abundance mRNAs

• New strategies for gene knockout

• Large-scale expression and purification of recombinant proteins in cultured cells

• High-throughput analysis of gene expression by real-time RT-PCR

• Gene expression profiling via DNA microarray

• Phage display

• siRNA technology

Each chapter covers the principles underlying the methods and techniques presented and a detailed step-by-step description of each protocol, as well as notes and tips. We have found that many of the currently available books in molecular biology contain only protocol recipes. Unfortunately, many fail to explain the principles and concepts behind the methods outlined or to inform the reader of possible pitfalls in the methods described. We intend to fill these gaps.

Although all four authors have worked as a team, for the information of the reader, the following table shows which authors wrote which chapters.

Chapter Number



W. Wu, P.B. Kaufman, M.J. Welsh

2, 3, 6, 10, 12-14

W. Wu

8, 9, 16, 17

W. Wu, M.J. Welsh

5, 7, 15

W. Wu, P.B. Kaufman

4, 11, 18

W. Wu, H.H. Zhang


W. Wu, M.J. Welsh, H.H. Zhang

William Wu Michael J. Welsh Peter B. Kaufman Helen H. Zhang

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