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Recombinant DNA Technology

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Presentation on theme: "Recombinant DNA Technology"— Presentation transcript:

1 Recombinant DNA Technology
Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus web site

2 Plasmids and other cloning vectors

3 7.1 DNA cloning with plasmid vectors
Recombinant DNA technology depends on the ability to produce large numbers of identical DNA molecules (clones) Clones are typically generated by placing a DNA fragment of interest into a vector DNA molecule, which can replicate in a host cell When a single vector containing a single DNA fragment is introduced into a host cell, large numbers of the fragment are reproduced along with the vector Two common vectors are E. coli plasmid vectors and bacteriophage  vectors Copyright (c) by W. H. Freeman and Company

4 7.1 Plasmids are extrachromosomal self-replicating DNA molecules
Figure 7-1 Copyright (c) by W. H. Freeman and Company

5 7.1 The general procedure for cloning with plasmid vectors
Figure 7-3 Copyright (c) by W. H. Freeman and Company

6 Copyright (c) by W. H. Freeman and Company
7.1 Plasmid cloning permits isolation of DNA fragments from complex mixtures Figure 7-4 Copyright (c) by W. H. Freeman and Company

7 Restriction enzymes and other cloning tools

8 7.1 Restriction enzymes cut DNA molecules at specific sequences
Figure 7-5a Copyright (c) by W. H. Freeman and Company

9 7.1 Restriction enzymes cut DNA molecules at specific sequences
Figure 7-5b Copyright (c) by W. H. Freeman and Company

10 7.1 Selected restriction enzymes
Copyright (c) by W. H. Freeman and Company

11 Copyright (c) by W. H. Freeman and Company
7.1 Restriction enzymes cut an organism’s DNA into a reproducible set of restriction fragments Figure 7-6 Copyright (c) by W. H. Freeman and Company

12 Copyright (c) by W. H. Freeman and Company
7.1 Restriction fragments with complementary “sticky ends” are ligated easily Figure 7-7 Copyright (c) by W. H. Freeman and Company

13 Copyright (c) by W. H. Freeman and Company
7.1 Polylinkers facilitate insertion of restriction fragments into plasmid vectors e.g. pBluescript (map on p. 10) Copyright (c) by W. H. Freeman and Company

14 7.1 Small DNA molecules can be chemically synthesized
Synthetic DNA is useful for: generating polylinker sequences, sequencing DNA, isolating clones of interest, creating site-specific mutations Copyright (c) by W. H. Freeman and Company Figure 7-9

15 Gene Libraries

16 7.2 Constructing DNA libraries with  phage and other cloning vectors
Cloning all of the genomic DNA of higher organisms into plasmid vectors is not practical due to the relatively low transformation efficiency of E. coli and the small number of transformed colonies that can be grown on a typical culture plate Cloning vectors derived from bacteriophage do not suffer from such limitations A collection of clones that includes all the DNA sequences of a given species is called a genomic library A genomic library can be screened for clones containing a sequence of interest Copyright (c) by W. H. Freeman and Company

17 7.2 The bacteriophage genome
Figure 7-10 Copyright (c) by W. H. Freeman and Company

18 Copyright (c) by W. H. Freeman and Company
7.2 Nearly complete genomic libraries of higher organisms can be prepared by  cloning Figure 7-12 Copyright (c) by W. H. Freeman and Company

19 Copyright (c) by W. H. Freeman and Company
7.2 Complementary DNA (cDNA) libraries are prepared from isolated mRNAs Figure 7-14 Copyright (c) by W. H. Freeman and Company

20 7.2 Preparation of a bacteriophage  cDNA library
Copyright (c) by W. H. Freeman and Company Figure 7-15

21 7.2 Larger DNA fragments can be cloned in cosmids and other vectors
Figure 7-16 Copyright (c) by W. H. Freeman and Company

22 Screening libraries to isolate genes

23 7.3 Identifying, analyzing, and sequencing cloned DNA
The most common approach to identifying a specific clone involves screening a library by hybridization with radioactively labeled DNA or RNA probes. Copyright (c) by W. H. Freeman and Company

24 7.3 The membrane-hybridization assay
Double stranded DNA Melt Single-stranded DNA DNA binds to filter Filter Incubate with labeled DNA Hybridized complemetary DNAs Wash away labeled DNA that did not hybridize to DAN bound to filter Figure 7-17 Copyright (c) by W. H. Freeman and Company Perform autoradiography

25 Copyright (c) by W. H. Freeman and Company
7.3 Identification of a specific clone from a  phage library by membrane hybridization Figure 7-18 Copyright (c) by W. H. Freeman and Company

26 Copyright (c) by W. H. Freeman and Company
7.3 Oligonucleotide probes are designed based on partial protein sequences Figure 7-19 Copyright (c) by W. H. Freeman and Company

27 Copyright (c) by W. H. Freeman and Company
7.3 Specific clones can be identified based on properties of the encoded proteins Copyright (c) by W. H. Freeman and Company Figure 7-21

28 Clone Characterizarion

29 7.3 Gel electrophoresis resolves DNA fragments of different size
Figure 7-22 Copyright (c) by W. H. Freeman and Company

30 Copyright (c) by W. H. Freeman and Company
7.3 Visualization of restriction fragments separated by gel electrophoresis Figure 7-23 Copyright (c) by W. H. Freeman and Company

31 7.3 Pulsed-field gel electrophoresis separates large DNA molecules
Figure 7-26 Copyright (c) by W. H. Freeman and Company

32 DNA sequencing techniques discussed in lecture 2
GenBank Sequence Database Link to miscellaneous genomics tools and databases Bioinformatics

33 Copyright (c) by W. H. Freeman and Company
7.4 Bioinformatics Bioinformatics is the rapidly developing area of computer science devoted to collecting, organizing, and analyzing DNA and protein sequences Using searches based on homologous sequences, stored sequences suggest functions of newly identified genes and proteins Homologous proteins involved in genetic information processing are widely distributed Copyright (c) by W. H. Freeman and Company

34 Copyright (c) by W. H. Freeman and Company
7.4 Comparative analysis of genomes reveals much about an organism’s biology Copyright (c) by W. H. Freeman and Company

35 Copyright (c) by W. H. Freeman and Company
7.4 The C. elegans genome encodes numerous proteins specific to multicellular organisms Copyright (c) by W. H. Freeman and Company

36 Analysis of genes and gene products

37 7.5 Analyzing specific nucleic acids in complex mixtures
A specific DNA sequence isolated by cloning can serve as a probe to detect the presence and the amounts of complementary nucleic acids in complex mixtures including total cellular DNA or RNA Copyright (c) by W. H. Freeman and Company

38 7.5 Southern blotting detects specific DNA fragments
Figure 7-32 Copyright (c) by W. H. Freeman and Company

39 7.5 Northern blotting detects specific mRNAs
Figure 7-33 Copyright (c) by W. H. Freeman and Company

40 7.8 DNA microarrays: analyzing genome-wide expression
DNA microarrays consist of thousands of individual gene sequences bound to closely spaced regions on the surface of a glass microscope slide DNA microarrays allow the simultaneous analysis of the expression of thousands of genes The combination of DNA microarray technology with genome sequencing projects enables scientists to analyze the complete transcriptional program of an organism during specific physiological response or developmental processes Copyright (c) by W. H. Freeman and Company

41 7.8 A yeast genome microarray
Figure 7-39 Copyright (c) by W. H. Freeman and Company

42 Protein Overexpression

43 7.6 Producing high levels of proteins from cloned cDNAs
Many proteins are normally expressed at very low concentrations within cells, which makes isolation of sufficient amounts for analysis difficult To overcome this problem, DNA expression vectors can be used to produce large amounts of full length proteins Copyright (c) by W. H. Freeman and Company

44 7.6 E. coli expression systems can produce full-length proteins
Figure 7-36 Copyright (c) by W. H. Freeman and Company

45 Copyright (c) by W. H. Freeman and Company
7.6 Even larger amounts of a desired protein can be expressed with a two-step system Figure 7-37 Copyright (c) by W. H. Freeman and Company

46


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