Lab Manual for Biomedical Research

A Rapid DNA Minipreparation Method Suitable for AFLP and Other PCR Applications - http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb17/17053-1.pdf
Preparation of DNA from plant tissues suitable for PCR methods including AFLP, article by DH CHEN and PC RONALD Department of Plant Pathology, University of California, Davis.

Adjuvants in PCR Reactions - http://info.med.yale.edu/genetics/ward/tavi/p16.html
Brief discussion of additives to improve amplification efficiency and specificity of PCR, by Octavian Henegariu, Yale-New Haven Medical Center.

Amberg Laboratory Protocols: - http://www.upstate.edu/biochem/amberg/protocols.php
Laboratory protocols for PCR work used by the group of David Amberg at the Department of Biochemistry and Molecular Biology Upstate Medical University, Syracuse, New York.

Anchor Probes for Comparative Mapping of Grass Species - http://greengenes.cit.cornell.edu/anchors/
Article in which probes from different libraries were used to hybridize seven cereals at the Department of Plant Breeding and Biometry, Cornell University, NY.

Attotron Biosensor Corporation - http://www.attotron.com
Research and development company for development of biosensors and related products for the research and educational markets.

BioRad, Amplification, PCR - http://www.bio-rad.com/B2B/BioRad/product/br_category.jsp?
Division of BioRad Laboratories that manufactures and sells instruments for PCR, in Hercules, California, USA.

Degenerate PCR - http://www.dartmouth.edu/~ambros/protocols/other/koelle/degenerate_PCR.html
The identification of novel members of gene families by PCR using degenerate primers is described and protocols given. Article by Michael Koelle 1996 on the web site of Dartmouth College.

Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences - http://www.uni-graz.at/~binder/thesis/node64.html
Thesis abstract about restriction fragment length polymorphism (RFLP) by Alexander Binder 1997.

Detection of Single Nucleotide Mutations in Wheat Using Single Strand Conformation Polymorphism Gels - http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb19/R01-013.pdf
P Martins-Lopez, H Zhang, R Koebner, Plant Mol. Biol. Reporter 19(2001): 159-162. From National Research Coouncil Canada.

DNALC: PCR Animation - http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html
An animation explaining how the Polymerase Chain Reaction (PCR) works, from the Dolan DNA learning center, Cold Spring Harbor Laboratory, USA.

Dolan DNA Learning Centers Gene Almanac - http://www.dnalc.org/home.html
Educational site on topics in genetics and gene expression from Cold Spring Harbor Laboratory, USA.

Effect of PCR Buffer on Multiplex PCR - http://www.qiagen.com/literature/brochures/pcr/pdf/pcrcha03.pdf
Multiplex PCR employs different primer pairs in the same amplification reaction. This requires extensive optimization of annealing conditions. From Quiagen (company).

Fidelity of DNA Polymerases for PCR - http://www.lecb.ncifcrf.gov/~pnh/papers/TIBS/aug95.html
Article by PN Hengen from TIBS 1995

FISH Guide and Troubleshooting - http://info.med.yale.edu/genetics/ward/tavi/FISHguide.html
Links to pages describing influential parameters, with guides on PCR, RT-PCR and multiplex PCR reactions, Taq, FISH, CM-FISH, TM-FISH, microarrays, CCK, slide prep and labeling, maintained by Octavian Henegariu from Yale University, New Haven, CT.

Fluorescence in Situ Hybridisation - http://info.med.yale.edu/genetics/ward/tavi/FISH.html
Technical notes on Fluorescence in situ hybridisation from the Institute of Genetics of Yale School of Medicine, New Haven, Connecticut, USA.

GeneOhm Sciences - http://www.geneohm.com
Tests on group B Streptococcus and methicillin resistant Staphylococcus aureus by PCR / DNA sequencing.

GenHunter - http://www.genhunter.com
Manufacturer of material for differential display PCR in Nashville, Tenn USA.

HiFi DNA - http://www.hifidna.com/
HiFi DNA is a company selling a DNA polymerase for PCR at low temperature giving accurate replication of certain sequences where Taq fails.

Ingenetix GmbH - http://www.ingenetix.com/
Develops technology and products for DNA and mRNA research. Also provide DNA testing for the determination of parentage/paternity and custom DNA sequencing, oligonucleotide synthesis, genotyping services, pharmacogenetics and quantitative PCR, in Vienna, Austria.

Inverse PCR and Cycle Sequencing of P Element Insertions for STS Generation - http://www.fruitfly.org/about/methods/inverse.pcr.html
Step by step protocol, by EJ Rehm, Berkeley Drosophila Genome Project, USA.

Inverse PCR for PAC-end Sequencing - http://www.genetics.wustl.edu/fish_lab/frank/cgi-bin/fish/prot2.html
To generate PCR fragments that contain the ends of PAC inserts that can be sequenced. Protocol by B Barbazuk, Washington University Zebrafish Genome Resources Project, USA.

Inverse PCR for Use with Snyder mTn-lacZ/LEU2-based Mutagenesis - http://labs.fhcrc.org/gottschling/General%20Protocols/ipcr.html
Protocol by M McMurray, Fred Hutchinson Cancer Research Center, Seattle, Wa. USA.

Inverse PCR Protocol - http://www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/inverse_pcr_protocol.html
Step by step protocol, from the web site of the Department of Biology, University of Michigan, USA.

Kary B. Mullis - Autobiography - http://nobelprize.org/chemistry/laureates/1993/mullis-autobio.html
The originator of PCR, from the Nobel e-museum web site.

Kary Mullis - http://www.invent.org/hall_of_fame/109.html
Inventor Profile of Kary Mullis, the originator of PCR, from the National Inventors Hall of Fame web site.

Long PCR Protocol - http://twod.med.harvard.edu/labgc/estep/longPCR_protocol.html
Protocol and guidelines for choice of conditions for PCR of long sequences (10 kb or larger). From Genetics Dept., Harvard Medical School, Boston, MA, USA

Nematode ITS1 Size Variation - http://nematode.unl.edu/its_id/EXAMPLES/index.htm
Examples of Restriction Fragment Length Polymorphism (RFLP)electrophoresis slabs for different nematodes, from University of Nebrasca.

Optimizing Multiplex and LA-PCR with Betaine - http://www-lecb.ncifcrf.gov/~pnh/papers/TIBS/jun97.html
LA-PCR = "long and accurate PCR". Article by PN Hengen in TIBS June 1997.

Optimizing PCR Protocols - http://www.jax.org/imr/optimize_pcr.html
Brief guidelines. From the Jackson Laboratory, University of Maine, USA.

PCR Amplification of cDNA Segments by 2 Stage Nested PCR - http://www.ncbi.nlm.nih.gov/SNP/snp_viewTable.cgi?type=method&method_id=555
Protocol from the method database of NIH, USA.

PCR and Multiplex PCR Guide - http://www.info.med.yale.edu/genetics/ward/tavi/Guide.html
Discussions of the parameters influencing the PCR reaction and some PCR and multiplex PCR applications, by Octavian Henegariu on the web site of the Yale - New Haven Medical center.

PCR Animated - http://users.ugent.be/~avierstr/principles/pcrani.html
Animation illustrating the principle of PCR, from the University of Ghent, Belgium.

PCR Gateway - http://www.horizonpress.com/pcr/
A directory of PCR techniques, PCR protocols, PCR troubleshooting, PCR websites and online resources from the publisher Horizon Press.

PCR Guru - http://www.pcrguru.com/
A downloadable textbook on PCR setup and optimization, not free.

PCR Method Protocols - http://hg.wustl.edu/hdk_lab_manual/pcr/pcrcontents.html
Protocols for PCR posted by the Helen Donis-Keller Laboratory.

PCR Primer Design and Reaction Optimisation - http://web.uct.ac.za/microbiology/pcroptim.htm
Article by Ed Rybicki, Department of Molecular and Cell Biology, University of Cape Town in: Molecular Biology Techniques Manual, on the web site of the University of Cape Town.

PCR Project - http://sunsite.berkeley.edu/biotech/pcr/
Presentations from the University of California at Berkley on PCR, both current research reports and reviews.

PCR Protocol - http://www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/general_pcr_protocol.html
Detailed PCR protocol from the web site of the Department of Biology, University of Michigan, USA.

PCR Protocols - http://www.cas.psu.edu/docs/CASDEPT/VET/jackvh/jvhpcr.html
Protocols and technical hints, particularly for reverse transcription PCR, somewhat outdated, compiled by Dr Jack Vanden Heuvel, Department of Veterinary Science and Molecular Toxicology Program, Penn State University

PCR Technology - http://www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.html
An introduction by Connie Veilleux from the US National Health Museum website.

PCR Troubleshooting - http://info.med.yale.edu/genetics/ward/tavi/Trblesht.html
Limited to conventional straight forward PCR. Page designed and maintained by Octavian Henegariu on the web site of the Yale - New Haven Medical Center.

PCR World - http://pcrworld.blogspot.com
Collection of unreferenced texts on various aspects of PCR.

PCR-ELISA and Related - http://www.btc-bti.com/pcrelisa.htm
P Zhang, CJ Gebhart, D Burden, GE Duhamel: A low technology alternative to real time PCR, technical article on the site of BT&C, Inc Bridgewater, NJ, USA.

Polymerase Chain Reaction - http://www.accessexcellence.com/RC/CT/polymerase_chain_reaction.html
Popular survey article by Mark V. Bloom, DNA Learning Center, Cold Spring Harbor Laboratory, from the web site of the US National Health Museum.

Polymerase Chain Reaction (PCR) - http://www.accessexcellence.org/AB/GG/polymerase.html
A graphic description of the principle of PCR from the US National Health Museum web site.

PrimerDigital - http://primerdigital.com/index.php
International biotechnology company specialized in a design service for PCR primers and probes, PCR-based technology development, projects for development of polymorphism and software development.

Primerfox - http://www.primerfox.com
Free online tool for generation of PCR primers.

Principle of PCR - http://users.ugent.be/~avierstr/principles/pcr.html
Applications in work on aging of Caenorhabditis elegans and phylogeny of nematodes, by Andy Vierstraete, Department of Biology, University of Ghent, Belgium.

Protocols Online: PCR Protocols - http://www.protocol-online.org/prot/Molecular_Biology/PCR/
Extensive collection of PCR protocols and methods from Protocol On Line.

Quantitative PCR Protocol - http://www.jax.org/cyto/quanpcr.html
From the Jackson Laboratory, University of Maine, USA.

RAPD PCR - http://avery.rutgers.edu/WSSP/StudentScholars/project/archives/onions/rapd.html
RAPD stands for Random Amplification of Polymorphic DNA, where the target sequence(s) (to be amplified) is unknown.Brief description, from Rutgers University, USA.

Rational Primer Design Greatly Improves Differential Display-PCR (DD-PCR) - http://nar.oxfordjournals.org/cgi/content/full/25/11/2239
Article: D Graf, AG Fisher, M Merkenschlager: Nucl. Acids Res. 25:11 2239-2240.

Reference in PCR - http://www.gene-quantification.org
Technical aspects of quantitative real-time PCR and RT-PCR. Instruments, kits, dyes, chemistries, and services presented by their manufacturers.

Rep-PCR Genomic Fingerprinting - http://www.msu.edu/user/debruijn/
Bacteria are characterized by Rep-PCR fingerprinting using primers corresponding to naturally occurring repetitive sequences in the interspersed regions.

RFLP Definition - http://vm.cfsan.fda.gov/~frf/rflp.html
RFLP = Restriction Fragment Length Polymorphism, from FDA

Roe Laboratory Protocols - http://www.genome.ou.edu/proto.html
Molecular biological protocols, mostly PCR related used by Bruce A. Roe at the Dept. of Chemistry and Biochemistry, OU, Norman, OK.

Single Tube Confirmation PCR Protocol - http://www-sequence.stanford.edu/group/yeast_deletion_project/single_tube_protocol.html
For characterization colonies of transformed clones of Saccharaomyces, from the web site of the Stanford Genome Technology Center, Palo Alto, CA, USA.

Standard PCR Protocols - http://web.uct.ac.za/microbiology/pcrcond.htm
From Molecular Biology Techniques Manual, from the web site of the University of Cape Town, South Africa.

T-DNA Generated Enhancer Traps in Arabidopsis - http://www.dartmouth.edu/~tjack/
Application of inverse PCR, partial genomic libraries and TAIL-PCR in cloning flanking, at the Department of Biological Sciences, Dartmouth College, Hanover, NH.

Tavi's PCR Protocols - http://info.med.yale.edu/genetics/ward/tavi/PCR.html
A page describing the main parameters and trouble-shooting in PCR. The page is somewhat dated (updated 1997) but still useful.

The PCR Encyclopedia - http://www.pcr-encyclopedia.com/
Describes plans for a free encyclopedia dedicated to the polymerase chain reaction (PCR).

The Web Guide of PCR - http://www.pcrlinks.com/
List of links and forum on the subject and related methodology. Set up and maintained by SJ Krivokapich, National University of Misiones, Argentina.

Thermostable DNA Polymerases - http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/hotpolys.html
Discussion of their origin and briefly their properties. From the web site of Colorado State University.

Wayward PCR Primers - http://www-lecb.ncifcrf.gov/~pnh/papers/TIBS/jan95.html
Article by PN Hengen from TIBS 1995 on the loss of activity of PCR primers with time.

What the Heck is PCR? - http://people.ku.edu/~jbrown/pcr.html
Popular description of the PCR technique by John C Brown, University of Kansas 1995.

Which DNA Marker for Which Purpose? - http://webdoc.sub.gwdg.de/ebook/y/1999/whichmarker
Compendia of the Research Project "Development, optimisation and validation of molecular tools for assessment of biodiversity in forest trees", European Union DGXII Biotechnology FW IV Research Programme. From the web site of the University Library, Göttingen.

Sunday, September 14, 2008

History of Polymerase chain reaction (PCR)--(1)

From Wikipedia, the free encyclopedia
The history of the Polymerase Chain Reaction (or PCR) has variously been described as a classic "Eureka!" moment[1], or as an example of cooperative teamwork between disparate researchers[2]. A list of some of the events before, during, and after its development:

Prelude

On April 25, 1953 James D. Watson and Francis Crick publish "a radically different structure" for DNA[3], thereby founding the field of Molecular Genetics. Their structure involves two strands of complementary base-paired DNA, running in opposite directions as a double helix. They conclude their report saying that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material". They are awarded the Nobel Prize in 1962.

Starting in the mid 1950s, Arthur Kornberg begins to study the mechanism of DNA replication[4]. By 1957 he has identified the first DNA polymerase[5]. The enzyme is surprisingly limited, creating DNA in just one direction and requiring an existing primer to initiate copying of the template strand. However, the overall DNA replication process is surprisingly complex, requiring separate proteins to open the DNA helix, to keep it open, to create primers, to synthesize new DNA, to remove the primers, and to tie the pieces all together. He is awarded the Nobel Prize in 1959.

In the early 1960s H. Gobind Khorana participates in the discovery of the Genetic Code. Afterwards, he initiates a large project to totally synthesize a functional human gene[6]. To achieve this, he pioneers many of the techniques needed to make and use synthetic DNA oligonucleotides. Sequence-specific oligos are used both as building blocks for the gene, and as primers and templates for DNA polymerase. In 1968 Khorana is awarded the Nobel Prize for his work on the Genetic Code.

In 1969 Thomas Brock reports the isolation of a new species of bacterium from a hot spring in Yellowstone National Park. Naming it Thermus aquaticus[7] (Taq), it goes on to become a standard source of enzymes able to withstand higher temperatures than those from E. Coli.

In 1970 a modified version of DNA Polymerase I from E. coli is reported[8]. Treatment with a protease removes the 'forward' nuclease activity of this enzyme. The overall activity of the resulting Klenow fragment is therefore biased towards the synthesis of DNA, rather than its degradation.

By 1971 researchers in Khorana's project, concerned over their yields of DNA, begin looking at "repair synthesis" - an artificial system of primers and templates that allows DNA polymerase to copy segments of the gene they are synthesizing. Although similar to PCR in using repeated applications of DNA polymerase, the process they usually describe[9] employs just a single primer-template complex, and therefore would not lead to the exponential amplification seen in PCR.

Also by 1971 Kjell Kleppe, a researcher in Khorana's lab, envisions a process very similar to PCR. At the end of a paper on the earlier technique[10], he describes how a two-primer system might lead to replication of a specific segment of DNA:

"... one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed
with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original
duplex should result. The whole cycle could be repeated, there being added every time a fresh dose of the enzyme." [10]

No results are shown there, and the mention of unpublished experiments in another paper[9] may (or may not) refer to the two-primer replication system. (These early precursors to PCR were carefully scrutinized in a patent lawsuit, and are discussed in Mullis' chapters in [11].)

Also in 1971, Cetus Corporation is founded in Berkeley, California by Ronald Cape, Peter Farley, and Donald Glaser. Initially the company screens for microorganisms capable of producing components used in the manufacture of food, chemicals, vaccines, or pharmaceuticals. After moving to nearby Emeryville, they take up projects involving the new biotechnology industry, primarily the cloning and expression of human genes, but also the development of diagnostic tests for genetic mutations.

In 1976 a DNA polymerase[12] is isolated from T. aquaticus. It is found to retain its activity at temperatures above 75°C.

In 1977 Frederick Sanger reports a method for determining the sequence of DNA[13]. The technique involves an oligonucleotide primer, DNA polymerase, and modified nucleotide precursors that block further extension of the primer in sequence-dependent manner. He is awarded the Nobel Prize in 1980.

Thus, by 1980 all of the components needed to perform PCR amplification were known to the scientific community. The use of DNA polymerase to extend oligonucleotide primers was a common procedure in DNA sequencing and the production of cDNA for cloning and expression. The use of DNA polymerase for nick translation was the most common method used to label DNA probes for Southern blotting.

Theme

In 1979 Cetus Corporation hires Kary Mullis to synthesize oligonucleotides for various research and development projects throughout the company[14]. These oligos are used as probes for screening cloned genes, as primers for DNA sequencing and cDNA synthesis, and as building blocks for gene construction. Originally synthesizing these oligos by hand, Mullis later evaluates early prototypes for automated synthesizers[1].

By May 1983 Mullis has synthesized oligo probes for a project at Cetus attempting to analyze a mutation for a human genetic disease. Hearing of problems with their work, Mullis envisions an alternative technique based on Sanger's DNA sequencing method[14]. Realizing the difficulty in making that method specific to a single location in the genome, Mullis considers adding a second primer on the opposite strand. He then generalizes the idea, and realizes that repeated applications of polymerase could lead to a chain reaction of replication for a specific segment of the genome - PCR.

Later in 1983 Mullis begins to test his idea. His first experiment[2] does not involve thermal cycling - he hopes that the polymerase can perform continued replication on its own. Later experiments that year do involve repeated thermal cycling, and target small segments of a cloned gene. Mullis considers these experiments a success, but is unable to convince other researchers.

In June 1984 Cetus holds its annual meeting in Monterey, California. Its scientists and consultants present their results, and consider future projects. Mullis presents a poster on the production of oligonucleotides by his laboratory, and shows some of the results from his experiments with PCR[2]. Only Joshua Lederberg, a Cetus consultant, shows any interest[14]. Later at the meeting, Mullis is involved in a physical altercation with another Cetus researcher, over a dispute unrelated to PCR[2]. The other scientist soon leaves the company, and Mullis is removed as head of the oligo synthesis lab. The days of his continued employment at Cetus may be numbered.

Development

In September of 1984 Tom White, VP of Research at Cetus (and a close friend), pressures Mullis to take his idea to the group developing the genetic mutation assay. Together, they spend the following months designing experiments that could convincingly show that PCR is working on genomic DNA. Unfortunately, the expected amplification product is not visible in agarose gel electrophoresis[15], leading to confusion as to whether the reaction has any specificity to the targeted region.

In November of 1984[2] the amplification products are analyzed by Southern blotting, which clearly shows an increasing amount of the expected 110 bp DNA product[16]. Having the first visible signal, the researchers are able to begin finding optimum conditions for the reaction. Later, the amplified products are cloned and sequenced, showing that only a small fraction of the amplified DNA is the desired target, and that the polymerase then being used only rarely incorporates incorrect nucleotides during replication[15].

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