problem with final exam and course grade
several students each term have decrease in exam scores:
99 - 92 - 73
94 - 72 - 88
99 - 99 - 95
overview...... LibraryOverview.gif
Palindromes ..(not on web)... palindromes.gif
RestrictionEnzymes.GIF
and methylation
"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"
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. 302)
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
Picture of pUC19..... plasmidpUC19.gif
which plasmid has foreign DNA?...plasmid1.gif
which bacteria have the plasmid? ...plasmid2.gif
which colony has the DNA we are looking for? ......plasmid3.gif
Review of insertion of foreign DNA into vector
Figure 14.5..(not on web).. 14x5.gif
Lambda vector ....... l
Figure 14.6 ... 14x6.gif
LambdaVector.gif
YAC - Yeast artificial chromosome
Figure 14.7 .. 14x7.gif
BAC - Bacterial artificial chromosome
Use of modified F plasmid in E. coli
review of illustrations in the text
insertion of foreign DNA into plasmid .... 14x5.gif
insertion of foreign DNA into lambda vector .... 14x6.gif
structure of yeast artificial chromosome (YAC) ....14x7.gif
construction of cDNA ... for library or probe .... 14x8.gif
attachment of sticky end to cDNA for cloning .... 14x9.gif
screening plasmid genomic library for specific DNA ..... 14x11a.gif .....14x11b.gif
construction of restriction map .... 14x13.gif
Southern blot to find DNA matching a cDNA probe .... 14x14.gif
SouthernBlot.gif
example (not on web) .... hbs.gif
Genomic libraries - entire genome -for 3,000,000 - Kb human genome, need 920,000 clones in lambda
Chromosome libraries - sort chromosomes, use digest of only one chromosome
cDNA libraries - use reverse transcriptase to get DNA complementary to mRNA
Concept of the probe .... ProbeTarget.html
Determination of which colony has the DNA wanted see Figure 14.11
14x11a.gif; .... 14x11b.gif
Gilbert and Sanger Nobel Prize in Chemistry 1980
Sanger method discussed in text.
Use of dideoxynucleotide DNA precursor. Figures 14.16 and 9.5... 9.5; 14.16.gif
Technique in Figure 14.15..... 14x15.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
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)
last week, just before final exam, a time of great stress.....
lambda vector 600,000 library human
YAC vector 12,000 library
Sequence of temperatures and events as in Text Figure 14.19 .... PCRasText2.gif
Graph fixed and variable 3' ends ..... PCRFixedVariable.gif
Simple PCR movie...... PCRmovie.html
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
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 Southern blotting
And, to chapter 15... Chapter15.html