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

Lecture 5

Recombinant DNA TechnologyCloning Vectors

Gene Libraries

Clone Identification and Characterization

Reading: Chapter 9

Molecular Biology syllabus web site

http://a32.lehman.cuny.edu/webwurtzel/course/CURRIC99j.htm

Plasmids and other cloning vectors

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


7.1 Plasmids are extrachromosomal self-replicating DNA molecules

Figure 7-1


7.1 The general procedure for cloning with plasmid vectors

Figure 7-3


7.1 Plasmid cloning permits isolation of DNA fragments from complex mixtures

Figure 7-4

Restriction enzymes and other cloning tools


7.1 Restriction enzymes cut DNA molecules at specific sequences

Figure 7-5a


7.1 Restriction enzymes cut DNA molecules at specific sequences

Figure 7-5b


7.1 Selected restriction enzymes


7.1 Restriction enzymes cut an organism’s DNA into a reproducible set of restriction fragments

Figure 7-6


7.1 Restriction fragments with complementary “sticky ends” are ligated easily

Figure 7-7


7.1 Polylinkers facilitate insertion of restriction fragments into plasmid vectors

e.g. pBluescript (map on p. 10)

http://www.stratagene.com/manuals/212205.pdf


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

Gene Libraries


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


7.2 The bacteriophage genome

Figure 7-10


7.2 Nearly complete genomic libraries of higher organisms can be prepared by cloning

Figure 7-12


7.2 Complementary DNA (cDNA) libraries are prepared from isolated mRNAs

Figure 7-14


7.2 Preparation of a bacteriophage cDNA library

Figure 7-15


7.2 Larger DNA fragments can be cloned in cosmids and other vectors

Figure 7-16

Screening libraries to isolate genes


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.


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


7.3 Identification of a specific clone from a phage library by membrane hybridization

Figure 7-18


7.3 Oligonucleotide probes are designed based on partial protein sequences

Figure 7-19


7.3 Specific clones can be identified based on properties of the encoded proteins

Figure 7-21

Clone Characterizarion


7.3 Gel electrophoresis resolves DNA fragments of different size

Figure 7-22


7.3 Visualization of restriction fragments separated by gel electrophoresis

Figure 7-23


7.3 Pulsed-field gel electrophoresis separates large DNA molecules

Figure 7-26

DNA sequencing

techniques discussed in lecture 2 GenBank Sequence Database

Link to miscellaneous genomics tools and databases

Bioinformatics

http://www.ncbi.nlm.nih.gov/

http://a32.lehman.cuny.edu/webwurtzel/CUNY%20Plant%20Sciences%20PhD%20Program/links.html

http://a32.lehman.cuny.edu/webwurtzel/CUNY%20Plant%20Sciences%20PhD%20Program/links.html


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


7.4 Comparative analysis of genomes reveals much about an organism’s biology


7.4 The C. elegans genome encodes numerous proteins specific to multicellular organisms

Analysis of genes and gene products


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


7.5 Southern blotting detects specific DNA fragments

Figure 7-32


7.5 Northern blotting detects specific mRNAs

Figure 7-33


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


7.8 A yeast genome microarray

Figure 7-39

Protein Overexpression


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


7.6 E. coli expression systems can produce full-length proteins

Figure 7-36


7.6 Even larger amounts of a desired protein can be expressed with a two-step system

Figure 7-37

Documents

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