Restriction enzymes pages 267-269
Cloning vectors and DNA cloning pages 269- 271
Recombinant DNA libraries pages 272- 274
DNA sequencing pages 279- 280
Polymerase chain reaction pages 281-283
"Bacterial plasmids are circular extrachromosomal elements that replicate autonomously within cells.."
"Plasmid cloning vectors are derivatives of these natural plasmids 'engineered' to have features useful for cloning DNA"
During last lecture we discussed restriction enzymes, vectors, and the insertion of foreign DNA into vectors as a way of making large quantities of the foreign DNA.
vectors and genome sizes
genomeandvector.GIF
the idea..From workbook page 42
Plasmid Foreign Sequence .... PlasmidForeignSeries.html
PlasmidForeign1.GIF
PlasmidForeign2.GIF
PlasmidForeign2a.GIF
PlasmidForeign3.GIF
Items needed for good vector (text p. 270)
a) origin sequence for replication in E. coli
b) unique restriction enzyme sites
c) selectable markers to identify
which E. coli colonies contain a vector and
which E. coli colonies with a vector have a vector with a DNA insert
Vector used in the text is pUC19
sequence plasmid vector ../PlasmidSequence.html
And, the concept of libraries - definitions from your text
genomic library: "A collection of molecular clones that contains at least one copy of every DNA sequence in a genome"
cDNA library: "The collection of molecular clones that contains cDNA copies of the entire mRNA population of a cell"
cDNA: "DNA copies made from an RNA template catalyzed by the enzyme reverse transcriptase"
kinds of DNA libraries
construction of cDNA ... for library or probe Figure 13.6 .... 13x6.gif
attachment of sticky end to cDNA for cloning Figure 13.7... 13x7.gif
libraries may be screened for specific DNA sequences Figure 13.8 screen sequence
replica plating - Joshua Lederberg - Lederberg, J. (1989). "Replica plating and indirect selection of bacterial mutants: isolation of preadaptive mutants in bacteria by sib selection." Genetics 121(3): 395-399.
by use of probes via radioactive isotope or florescent dye
Concept of the probe .... ProbeTarget.html
Gilbert and Sanger Nobel Prize in Chemistry 1980
Sanger method discussed in text.
Use of dideoxynucleotide DNA precursor. Figures 13.12 and 8.5... 13.12.gif
Technique in Figure 13.11..... 13x11.gif
Step by step of Sanger technique:
As a series of slides ......SangerSeqsSeries.html
Denature DNA, add primer.....SangerSequencing1.gif
plus dideoxy / adenine..............SangerSequencing2.gif
plus dideoxy / guanine .............SangerSequencing3.gif
plus dideoxy / cytosine ............SangerSequencing4.gif
plus dideoxy/ thymine ............SangerSequencing5.gif
all together on a gel ..............SangerSequencing6.gif
Free books at noon; 416 SB2
I am the secretary at the Portland Pre-Health society on campus and we are having our third member meeting on December 1st at SMSU 327 from 2.00-3.00 pm. I was wondering if you can announce this before the genetic class on Monday November 29th. Here are some information about the event.
Meeting will consist of three speakers : A speaker from Osteopathy medicine, A speaker from OHSU talking about phamacology and a speaker talking about general pre-health volunteer opportunities. All the pre-Health students are encouraged to attend and every one who attend will receive free pizza and beaverages.
The plan: Today PCR and operon; Wednesday- some aspects of eukaryotic gene regulation. and Friday , review of the course and course evaluation.
We have now looked at some of the techniques for amplifying DNA via vector cloning.
Whole genome, specific chromosome, and cDNA may be cloned.
These large quantities of DNA may be sequenced via the Sanger or modified Sanger methods.
Another way to amplify DNA is the PCR technique.
ways of making lots of a specific sequence of DNA
the ultimate: PCR
1993 Nobel Prize Chemistry
Picture... Mullus-1993-1.gif
also ..../Mullis.gif..
reference: Kary Mullis article in Scientific American April 1990
"....revelation came to me one Friday night in April, 1983, as I gripped the steering wheel of my car and snaked along a moonlit mountain road into northern California's redwood country." .....p56
"You're not going to believe this," I crowed. "It's incredible."
Lederberg comment: --- anyone could have thought of this
Mullis response
to make lots of DNA....from article (not on web)
Simple PCR movie...... PCRmovie.html
Sequence of temperatures and events as in Text Figure 13.14 .... PCRasText2.gif
Graph fixed and variable 3' ends ..... PCRFixedVariable.gif
PCR made possible by use of a temperature resistant DNA polymerase: from the bacterium Thermus aquaticus , Taq polymerase
functions at a high temperature, not denatured by temperature of denaturation (72º C is optimum temp)
Thermal cycler..(not on web). thermalcycler.gif
The process is patented. Here are some statements from a recent Carolina Biological Company catalog: "Practice of the patented Polymerase Chain Reaction (PCR) requires a license. The licensing covers both thermal cyclers and reagents. Manufacturers of thermal cyclers or reagents may have their products licensed for use in PCR by paying licensing fees to Hoffman-LaRoche, Inc. and Perkin Elmer Corporation, who own the patent rights to PCR."
Kinds of DNA amplification
cloning - large pieces, slow, libraries - use of vectors: plasmids, phage, BAC, YAC
PCR - fast, needs primers, small pieces of DNA
The use of PCR and cloning in forensic science, anthropology, studies of evolution
genomic and mitochondrial DNA from Romanov bones ... http://users.rcn.com/web-czar/dna.htm
and, New Yorker magazine 21/28 Aug 95
and, recent picture and comments, Science 306:407; 15 October 2004 ../RomanovFamily.gif
hybridization DNA/DNA and DNA/RNA; the power of base pairing!
denaturation of DNA to single strands
use of probes to identify large pieces of DNA
restriction enzymes to cut DNA at specific sites
vectors - plasmids, phage, artificial chromosomes of yeast and bacteria
introduction of foreign DNA into vectors (transformation for bacteria)
transfer of bacteria/DNA to nitrocellulose filter
replicate plating - make a copy of a pattern of bacterial colonies
in vitro synthesis of DNA to make primers
electrophoresis
And, to chapter 16... Chapter16.html