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

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Presentation on theme: "Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus."— Presentation transcript:

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

2 Plasmids and other cloning vectors

3 Copyright (c) by W. H. Freeman and Company 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

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

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

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

7 Restriction enzymes and other cloning tools

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

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

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

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

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

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)pBluescript

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

15 Gene Libraries

16 Copyright (c) by W. H. Freeman and Company 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

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

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

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

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

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

22 Screening libraries to isolate genes

23 Copyright (c) by W. H. Freeman and Company 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.

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

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

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

28 Clone Characterizarion

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

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

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

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

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

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

36 Analysis of genes and gene products

37 Copyright (c) by W. H. Freeman and Company 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

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

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

40 Copyright (c) by W. H. Freeman and Company 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

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

42 Protein Overexpression

43 Copyright (c) by W. H. Freeman and Company 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

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

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

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