Recombinant DNA Technology PLASMID VECTORS. Cloning into a Plasmid.
Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and...
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Transcript of Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and...
![Page 1: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/1.jpg)
Lecture 5
Recombinant DNA TechnologyCloning Vectors
Gene Libraries
Clone Identification and Characterization
Reading: Chapter 9
Molecular Biology syllabus web site
![Page 2: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/2.jpg)
Plasmids and other cloning vectors
![Page 3: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/3.jpg)
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
![Page 4: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/4.jpg)
Copyright (c) by W. H. Freeman and Company
7.1 Plasmids are extrachromosomal self-replicating DNA molecules
Figure 7-1
![Page 5: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/5.jpg)
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7.1 The general procedure for cloning with plasmid vectors
Figure 7-3
![Page 6: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/6.jpg)
Copyright (c) by W. H. Freeman and Company
7.1 Plasmid cloning permits isolation of DNA fragments from complex mixtures
Figure 7-4
![Page 7: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/7.jpg)
Restriction enzymes and other cloning tools
![Page 8: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/8.jpg)
Copyright (c) by W. H. Freeman and Company
7.1 Restriction enzymes cut DNA molecules at specific sequences
Figure 7-5a
![Page 9: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/9.jpg)
Copyright (c) by W. H. Freeman and Company
7.1 Restriction enzymes cut DNA molecules at specific sequences
Figure 7-5b
![Page 10: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/10.jpg)
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7.1 Selected restriction enzymes
![Page 11: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/11.jpg)
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7.1 Restriction enzymes cut an organism’s DNA into a reproducible set of restriction fragments
Figure 7-6
![Page 12: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/12.jpg)
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7.1 Restriction fragments with complementary “sticky ends” are ligated easily
Figure 7-7
![Page 13: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/13.jpg)
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)
![Page 14: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/14.jpg)
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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
![Page 15: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/15.jpg)
Gene Libraries
![Page 16: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/16.jpg)
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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
![Page 17: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/17.jpg)
Copyright (c) by W. H. Freeman and Company
7.2 The bacteriophage genome
Figure 7-10
![Page 18: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/18.jpg)
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7.2 Nearly complete genomic libraries of higher organisms can be prepared by cloning
Figure 7-12
![Page 19: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/19.jpg)
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7.2 Complementary DNA (cDNA) libraries are prepared from isolated mRNAs
Figure 7-14
![Page 20: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/20.jpg)
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7.2 Preparation of a bacteriophage cDNA library
Figure 7-15
![Page 21: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/21.jpg)
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7.2 Larger DNA fragments can be cloned in cosmids and other vectors
Figure 7-16
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Screening libraries to isolate genes
![Page 23: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/23.jpg)
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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.
![Page 24: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/24.jpg)
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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
![Page 25: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/25.jpg)
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7.3 Identification of a specific clone from a phage library by membrane hybridization
Figure 7-18
![Page 26: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/26.jpg)
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7.3 Oligonucleotide probes are designed based on partial protein sequences
Figure 7-19
![Page 27: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/27.jpg)
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7.3 Specific clones can be identified based on properties of the encoded proteins
Figure 7-21
![Page 28: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/28.jpg)
Clone Characterizarion
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7.3 Gel electrophoresis resolves DNA fragments of different size
Figure 7-22
![Page 30: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/30.jpg)
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7.3 Visualization of restriction fragments separated by gel electrophoresis
Figure 7-23
![Page 31: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/31.jpg)
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7.3 Pulsed-field gel electrophoresis separates large DNA molecules
Figure 7-26
![Page 32: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/32.jpg)
DNA sequencing
techniques discussed in lecture 2 GenBank Sequence Database
Link to miscellaneous genomics tools and databases
Bioinformatics
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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
![Page 34: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/34.jpg)
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7.4 Comparative analysis of genomes reveals much about an organism’s biology
![Page 35: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/35.jpg)
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7.4 The C. elegans genome encodes numerous proteins specific to multicellular organisms
![Page 36: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/36.jpg)
Analysis of genes and gene products
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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
![Page 38: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/38.jpg)
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7.5 Southern blotting detects specific DNA fragments
Figure 7-32
![Page 39: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/39.jpg)
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7.5 Northern blotting detects specific mRNAs
Figure 7-33
![Page 40: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/40.jpg)
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
![Page 41: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/41.jpg)
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7.8 A yeast genome microarray
Figure 7-39
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Protein Overexpression
![Page 43: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/43.jpg)
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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
![Page 44: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/44.jpg)
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7.6 E. coli expression systems can produce full-length proteins
Figure 7-36
![Page 45: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/45.jpg)
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7.6 Even larger amounts of a desired protein can be expressed with a two-step system
Figure 7-37
![Page 46: Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.](https://reader035.fdocuments.in/reader035/viewer/2022062404/5519d3a3550346695e8b4d8d/html5/thumbnails/46.jpg)