Tremendous strides in decoding the human genome have been made over the last ten years. Large, chromosome-scale, genome-scale EST-sequencing and mapping techniques have dramatically expanded the base of identified genes and sequenced DNA. Gene isolation and cloning techniques that emerged earlier on, however, still remain effective mainstays for constructing dense transcript maps to isolate coding sequences and disease-associated genes contained within a specific chromosomal region. Positional Cloning by Exon Trapping and cDNA Selection examines two powerful methods for locating the coding region of a given gene by isolating gene fragments from individual clones or pools of genomic clones. This comprehensive guide details the exon-trapping and cDNA selection processes step by step-from isolating genomic templates and nuclear splicing, to verifying generated clones and sequenced data, to analyzing exon libraries and cDNA sublibraries. Procedures covered include: * Exon-trapping systems and descriptions of pSPL1 and pSPL3 vectors. * Exon amplification protocols for preparing vectors for cloning; subcloning genomic DNA into vectors; transformation, analysis, and transfection of sublibraries; RNA transcription; and PCR amplification and PCR product cloning. * Evaluation of exon libraries by PCR colony testing, identifying artifactual clones using Southern blot, and sequencing and mapping back candidate exons. * Isolation of genomic templates, such as COSMID-, P1-, PAC, and YAC DNA. * cDNA selection experiment protocols including cDNA screening, hybridization, and biotinylation; preparation of genomic and cDNA sources; and PCR cloning. * Clone analysis and analysis by hybridization. Positional Cloning by Exon Trapping and cDNA Selection offers researchers, scientists, and graduate students an invaluable tool for probing gene distribution and molecular organization. Most importantly, it provides a critical approach to isolating specific disease genes within a targeted genomic area.

Bernhard Korn and Marie-Laure Yaspo are the authors of Positional Cloning by Exon Trapping and cDNA Selection, published by Wiley.

Preface

ix

Exon Amplification

1

(32)

Introduction

1

(5)

The Eukaryotic Nuclear Splicing Mechanism

1

(2)

Exon Trapping Systems

3

(1)

Vectors Allowing the Trapping of Internal Exons

3

(1)

Vectors Allowing the Trapping of 3' Terminal Exons

4

(1)

Choice of the Exon Trap Vector

4

(1)

Description of the pSPL1 Vector and Its Modified Version, pSPL3

5

(1)

Overview of the Exon Amplification Method

6

(2)

Summary of the Procedure (Figure 1.2)

6

(1)

List of Components Necessary for the Exon Amplification Experiment

7

(1)

Exon Amplification Protocol

8

(17)

Preparation of the Linearized pSPL3 Vector for Cloning

8

(1)

Linearization

8

(1)

Dephosphorylation

9

(1)

Verification of the Linearized Phosphatased Vector (L/P pSPL3)

9

(1)

Subcloning Genomic DNA into the pSPL3 Vector

10

(1)

Choice and Preparation of the Genomic Target DNA

10

(1)

Digestion of the Target Genomic DNA to Be Inserted into pSPL3

11

(1)

Ligation into the pSPL3 Vector

11

(1)

Transformation of the Sublibrary

12

(1)

Preparation of Electrocompetent Cells

12

(1)

Electroporation

12

(1)

Analysis of the Sublibrary in the pSPL3 Vector

13

(1)

Preparation of the Pooled Sublibrary DNA for Transfection

14

(1)

Transfection of the Sublibrary into Eukaryotic COS-7 Cells

15

(1)

Propagation of COS-7 Cells

15

(1)

Transfection of COS-7 Cells by Electroporation

15

(2)

RNA Preparation

17

(2)

Reverse Transcription: First-Strand cDNA Synthesis

19

(1)

Exon Amplification

19

(1)

Primary PCR Amplification

20

(1)

Treatment of the First PCR and Digestion with BstXI

21

(1)

Secondary PCR Amplification

21

(1)

Tertiary PCR Amplification

22

(1)

Cloning the PCR Products

23

(1)

Chromaspin 100 Column DNA Purification of the PCR Product

23

(1)

Cloning in the pAMP1 Vector

24

(1)

Analysis of the Exon Library

25

(5)

Evaluation of the Library by PCR Colony Testing

25

(1)

Identifying the artifactual Clones by Southern Blotting

26

(1)

Mapping Back Candidate Exons

27

(1)

Protocol for Radiolabelling Exons

27

(1)

Sequencing the Exons

28

(1)

Analysis of the Sequence Data and Identifying Genes

28

(2)

Permanent Storage of the Exon Library

30

(1)

Analysis of the Exon Library by a Strategy of Sampling Without Replacement

31

(2)

Preparation of Library Filters for Screening Purposes

31

(1)

Hybridizations

32

(1)

cDNA Selection

33

(26)

Introduction

33

(6)

Conventional cDNA Screening

33

(1)

Screening of Large Genomic Regions

33

(1)

The Complexity of the Source

33

(1)

Hybridization in Solution

34

(1)

Biotinylation

35

(1)

Source of Genomic DNA

36

(1)

Source of cDNA

36

(1)

Blocking, Hybridization, and Elution

37

(1)

Analysis of cDNA Sublibraries

38

(1)

Conclusion and Outlook

38

(1)

Genomic Templates

39

(2)

Cosmid, P1, and PAC DNA Isolation

39

(1)

YAC DNA Isolation

40

(1)

Competition DNA

41

(3)

Repetitive Sequences

41

(1)

Vector DNA

41

(1)

Host DNA

42

(1)

Preparation of Yeast Genomic DNA

42

(1)

Preparation of E. coli Genomic DNA

42

(1)

Fragmentation of Competition DNA

43

(1)

cDNA Source

44

(11)

Biotinylation

44

(1)

Competition of Genomic Template DNA

45

(1)

First Round of cDNA Selection

45

(1)

Hybridization in Liquid (1)

45

(1)

Immobilization of Genomic Templates, Wash, and Elution

46

(1)

Size Selection of Eluted cDNA

47

(1)

Buffer Exchange of Chromaspin400 Column

48

(1)

PCR of First Round of cDNA Selection

48

(1)

Analyze PCR Products on Gel

49

(1)

Second Round of cDNA Selection

50

(1)

Hybridization in Liquid (2): Wash and Elution of cDNA

50

(1)

PCR of the Second Round of cDNA Selection

51

(1)

Analyze PCR Products on Gel

51

(1)

Size Selection of Amplified cDNA

52

(1)

Analyze PCR Products on Gel

52

(1)

Cloning of PCR Products

53

(1)

Annealing of PCR Product with pAMP

53

(1)

Transformation

53

(1)

Preparation of CaCl2 Competent Cells

54

(1)

Transformation with CaCl2 Competent Cells

54

(1)

Clone Analysis

55

(1)

Individual Clones

55

(1)

Evaluation of cDNA Insert Size

55

(1)

Determination of Sequence

56

(1)

cDNA Sublibrary

56

(1)

Analysis by Hybridization

56

(3)

Random Oligonucleotide Labelling

56

(1)

Removal of Unincorporated Nucleotides

57

(1)

Competition of Radioactive Probe

57

(1)

Hybridization and Wash

57

(2)

Maps, Materials, and Equipment Specifications

59

(16)

Map of pSPL3

59

(1)

The CloneAmp System

60

(7)

Alkaline Lysis DNA Preparation

67

(1)

Alkaline Lysis Solutions

67

(1)

Procedure

67

(1)

Materials

68

(7)

Oligonucleotides

68

(1)

Buffers and Solutions

69

(6)

References

75

(2)

Index

77

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