Transcript Lecture 14 student powerpoint

Lecture 14 – Gene
Cloning
Based on Chapter 08 Genomics: The Mapping
and Sequencing of Genomes
Copyright © 2010 Pearson Education Inc.
1. Introduction
2. DNA Cloning
3. Restriction Enzymes
4. General Properties of Restriction Enzymes
5. Frequency of Occurrence of Restriction
Sites in DNA
6. Restriction Sties (examples)
7. Creation of Recombinant DNA Molecules
8. Cloning Vectors – Plasmid Cloning Vectors
9. Inserting DNA into a Cloning Vector
Types of Vectors
Thus far we’ve talked about simple cloning vectors – e.g.
pUC19, pBluescript
Other vectors that are useful for analysis of cloned DNA:
1. Shuttle Vectors – e.g. pRS426 et al.
2. Expression Vectors
3. PCR Cloning Vectors
4. Transcribable Vecotrs
5. Non-plasmid Vectors
6. BACs
7. YACs
Shuttle Vectors
Yeast Ura3 promoter
Yeast 2m ARS
E.coli ampr
promoter
Yeast Ura3 coding sequence
(selectable marker)
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pRS426 GPD
5726 bp
E.coli F1 ORI
(single copy)
E.coli ampr
Coding sequence
Yeast 3’ terminator
Multiple Cloning Site
Yeast GPD Promoter
E.coli ORI (multicopy)
NgoMIV
NseI
Acc651
KpnI
XhoI
SalI
ClaI
HindIII
XmaI
Expression Vectors
Practical Issues for Constructing Clones Using an
Expression Vector
Transcribable/Translatable Vectors
An Alternative to Molecular Cloning:
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction
PCR Cloning Vectors
+
DNA Ligase
Non-Plasmid Vectors
10. Artificial Chromosomes
11. Bacterial Artificial Chromosomes (BACs)
12.
Yeast Artificial Chromosomes (YACs)
13. Genomic Libraries
Cloning a Specific Gene
•
Cloning genes from organisms with sequenced
genomes usually involves designing PCR primers to
amplify the gene from either genomic or cDNA.
– The PCR product is then cloned using a PCR
Vector.
• To clone a gene from an organism that lacks a
sequenced genome, several approaches may be used.
•
The method selected will depend on the molecular
tools available.
Finding a Specific Clone Using a cDNA Library
Screening a Genomic Library
Comparing the cDNA Clone and Genomic
Clones
DNA
Primary RNA
Transcript
mRNA (cDNA)
Identifying Genes in Libraries by
Complementation of Mutations