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
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4. 7.1 Plasmids are extrachromosomal self-replicating DNA molecules
Figure 7-1
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5. 7.1 The general procedure for cloning with plasmid vectors
Figure 7-3
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7.1 Plasmid cloning permits isolation of DNA fragments from complex mixtures
Figure 7-4
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7. Restriction enzymes and other cloning tools

8. 7.1 Restriction enzymes cut DNA molecules at specific sequences
Figure 7-5a
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9. 7.1 Restriction enzymes cut DNA molecules at specific sequences
Figure 7-5b
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10. 7.1 Selected restriction enzymes
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7.1 Restriction enzymes cut an organism’s DNA into a reproducible set of restriction fragments
Figure 7-6
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7.1 Restriction fragments with complementary “sticky ends” are ligated easily
Figure 7-7
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7.1 Polylinkers facilitate insertion of restriction fragments into plasmid vectors
e.g. pBluescript (map on p. 10)
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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
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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
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17. 7.2 The bacteriophage genome
Figure 7-10
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7.2 Nearly complete genomic libraries of higher organisms can be prepared by  cloning
Figure 7-12
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7.2 Complementary DNA (cDNA) libraries are prepared from isolated mRNAs
Figure 7-14
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20. 7.2 Preparation of a bacteriophage  cDNA library
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Figure 7-15

21. 7.2 Larger DNA fragments can be cloned in cosmids and other vectors
Figure 7-16
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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.
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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
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Perform autoradiography

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7.3 Identification of a specific clone from a  phage library by membrane hybridization
Figure 7-18
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7.3 Oligonucleotide probes are designed based on partial protein sequences
Figure 7-19
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7.3 Specific clones can be identified based on properties of the encoded proteins
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Figure 7-21

28. Clone Characterizarion

29. 7.3 Gel electrophoresis resolves DNA fragments of different size
Figure 7-22
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7.3 Visualization of restriction fragments separated by gel electrophoresis
Figure 7-23
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31. 7.3 Pulsed-field gel electrophoresis separates large DNA molecules
Figure 7-26
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32. 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
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7.4 Comparative analysis of genomes reveals much about an organism’s biology
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7.4 The C. elegans genome encodes numerous proteins specific to multicellular organisms
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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
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38. 7.5 Southern blotting detects specific DNA fragments
Figure 7-32
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39. 7.5 Northern blotting detects specific mRNAs
Figure 7-33
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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
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41. 7.8 A yeast genome microarray
Figure 7-39
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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
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44. 7.6 E. coli expression systems can produce full-length proteins
Figure 7-36
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7.6 Even larger amounts of a desired protein can be expressed with a two-step system
Figure 7-37
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