Polymerase chain reaction

Priming Is Required to Start DNA Synthesis

• All DNA polymerases require a 3′–OH priming end toinitiate DNA synthesis.

Priming Is Required to Start DNA Synthesis

• The priming end can beprovided by an RNA primer, anick in DNA, or a primingprotein.

Taq polymerase

• Taq polymerase (pronounced; tæ k pɑ.lɪ.mɚ.ɛs) is a thermostable DNA polymerasenamed after the thermophilic bacterium Thermus aquaticus from which it wasoriginally isolated by Thomas D. Brock in 1965[1]. It is often abbreviated to "TaqPol" (or simply "Taq"), and is frequently used in polymerase chain reaction (PCR),methods for greatly amplifying short segments of DNA.

In 1989 Science Magazine named Taq polymerase its first "Molecule of the Year". Kary Mullis received the Nobel Prize in 1993, the only one awarded for research performed at a biotechnology company. By the early 1990s the PCR technique with Taq polymerase was being used in many areas, including basic molecular biology research, clinical testing, and forensics.

Hoffmann-La Roche eventually bought the PCR and Taq patents from Cetus for $330 million, from which it may have received up to $2 billion in royalties

http://en.wikipedia.org/wiki/DNA_polymerasehttp://en.wikipedia.org/wiki/Thermophilichttp://en.wikipedia.org/wiki/Thermus_aquaticushttp://en.wikipedia.org/wiki/Thomas_D._Brockhttp://en.wikipedia.org/wiki/Polymerase_chain_reactionhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/File:Kary_Mullis.jpghttp://en.wikipedia.org/wiki/Science_(journal)http://en.wikipedia.org/wiki/Nobel_Prize_in_Chemistryhttp://en.wikipedia.org/wiki/Biotechnologyhttp://en.wikipedia.org/wiki/Genetic_testinghttp://en.wikipedia.org/wiki/Forensicshttp://en.wikipedia.org/wiki/Hoffmann-La_Rochehttp://en.wikipedia.org/wiki/Hoffmann-La_Rochehttp://en.wikipedia.org/wiki/Hoffmann-La_Rochehttp://en.wikipedia.org/wiki/Patent

Thermus aquaticus

When studies of biological organisms in hot springs began in the 1960s, scientists thought that the life of thermophilic bacteria could not be sustained in temperatures above about 55° Celsius (131° Fahrenheit).[2] Soon, however, it was discovered that many bacteria in different springs not only survived but also thrived in higher temperatures. In 1969, Thomas D. Brock and Hudson Freeze of Indiana University reported a new species of thermophilic bacterium which they named Thermus aquaticus.[3] The bacterium was first discovered in the Lower Geyser Basin of Yellowstone National Park, near the major geysers Great Fountain Geyser and White Dome Geyser,[4] and has since been found in similar thermal habitats around the world.

It thrives at 70°C (160°F), but can survive at temperatures of 50°C to 80°C (120°F to 175°F).

http://en.wikipedia.org/wiki/File:Grand_prismatic_spring.jpghttp://upload.wikimedia.org/wikipedia/commons/4/48/Thermus_aquaticus.JPGhttp://en.wikipedia.org/wiki/Thermophilic_bacteriahttp://en.wikipedia.org/wiki/Thomas_D._Brockhttp://en.wikipedia.org/wiki/Indiana_University_Bloomingtonhttp://en.wikipedia.org/wiki/Indiana_University_Bloomingtonhttp://en.wikipedia.org/wiki/Specieshttp://en.wikipedia.org/wiki/Thermophilehttp://en.wikipedia.org/wiki/Bacteriumhttp://en.wikipedia.org/wiki/Geothermal_areas_of_Yellowstone#Upper_Geyser_Basinhttp://en.wikipedia.org/wiki/Yellowstone_National_Parkhttp://en.wikipedia.org/wiki/Geyserhttp://en.wikipedia.org/wiki/Great_Fountain_Geyserhttp://en.wikipedia.org/wiki/White_Dome_Geyserhttp://en.wikipedia.org/wiki/White_Dome_Geyser

Real time qPCR

http://www.google.co.kr/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=AS5B8hm4Q6QbwM&tbnid=yJLq3oQ0i2IsGM:&ved=0CAUQjRw&url=http://www.asuragen.com/Services/services/gene_expression/ab_taqman.aspx&ei=PYlbUeXVN4f_lAXTnYGQCA&bvm=bv.44697112,d.dGI&psig=AFQjCNFjeeJO_JgZNhSHeDTB2b85-5GNhQ&ust=1365039800645484

Jurassic Park in 1993

http://www.google.co.kr/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=nC7Dpa6sj9h__M&tbnid=LtYOEFI8SEA7uM:&ved=0CAUQjRw&url=http://www.scienceclarified.com/Ex-Ga/Fossil-and-Fossilization.html&ei=XIVbUbKWNdC2lQWL4IDoBQ&bvm=bv.44697112,d.dGI&psig=AFQjCNG2T6HHnoO3bV3fDD0pAke5PKlZCg&ust=1365038787917244

Synthesized chromosome DNA (Mycoplasma mycoides,) 100 mega. DNA was ‘programmed’ base pair by base pair

Empty bacteria Mycoplasma capricolum

Mycoplasma genitalium JCVI-1.0.

http://en.wikipedia.org/wiki/Mycoplasma_mycoideshttp://en.wikipedia.org/wiki/Mycoplasma_mycoideshttp://en.wikipedia.org/wiki/Mycoplasma_mycoideshttp://en.wikipedia.org/wiki/Mycoplasma_mycoideshttp://en.wikipedia.org/wiki/Mycoplasma_mycoideshttp://en.wikipedia.org/wiki/Mycoplasma_capricolumhttp://en.wikipedia.org/wiki/Mycoplasma_capricolum

DNA sequence

• From the DNA sequence it might bepossible to identify the genes present inthe DNA molecule, and possibly todeduce the functions of those genes.

DNA sequence

Polyacrylamide gel

Polyacrylamide gel electrophoresis: DNA Size dependent separation

Chain-termination methods

The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length. The newly synthesized and labeled DNA fragments are heat denatured, and separated by size (with a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence.

http://en.wikipedia.org/wiki/Primer_(molecular_biology)http://en.wikipedia.org/wiki/DNA_polymerasehttp://en.wikipedia.org/wiki/Fluorescencehttp://en.wikipedia.org/wiki/Deoxynucleotideshttp://en.wikipedia.org/wiki/DNA_polymerasehttp://en.wikipedia.org/wiki/Dideoxynucleotideshttp://en.wikipedia.org/wiki/Hydroxylhttp://en.wikipedia.org/wiki/Phosphodiester_bondhttp://en.wikipedia.org/wiki/DNA_denaturationhttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Polyacrylamide_gelhttp://en.wikipedia.org/wiki/Autoradiographyhttp://en.wikipedia.org/wiki/Radiographyhttp://en.wikipedia.org/wiki/Radiographyhttp://en.wikipedia.org/wiki/Radiography

• The sequence is obtained by making copies of a clonedDNA fragment in a way that enables the position ofeach of the four nucleotides to be identified

• Chain termination sequencing makes use of a purifiedDNA polymerase, the type of enzyme that makescopies of DNA molecule

Chain-termination methods

1) A short piece of DNA called anoligonucleotide (primer) is then attachedto DNA.

2) The new strand is made by the DNApolymerase and extends over the clonedfragment

3) DNA polymerase can makepolynucleotides that are severalthousand nucleotides in length, but in asequencing experiment synthesis of thenew strand is terminated prenatally wheneach of four dideoxynucleotides is added

4) Once incorporated a dideoxynucleotideblocks further elongation because itlacks the 3’-OH group needed to form aconnection with the next nucleotide

http://upload.wikimedia.org/wikipedia/commons/c/cb/Sequencing.jpghttp://www.google.co.kr/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=D2KMX-cjCEdtWM&tbnid=xwPwRfBFLrzovM:&ved=0CAUQjRw&url=http://biocadmin.otago.ac.nz/fmi/xsl/bioc2h/learnbitslecture.xsl?-db=bioc2hweb.fp7&-lay=Lectures&-recid=4604&-find=&ei=rCJZUZf4Jo6pkgXGs4CQBQ&bvm=bv.44442042,d.dGI&psig=AFQjCNHLOpZRLwRWv0XI9b6VKpRjSjWisQ&ust=1364882442050266

ddA ddG

ddC ddT

http://www.google.co.kr/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=_5rqzmvJfvYBaM&tbnid=5QebqBOQ7j-CAM:&ved=0CAUQjRw&url=http://temasbiologiamolecular.blogspot.com/2012/05/secuenciacion-de-acidos-nucleicos.html&ei=HSJZUaWMN4WhkQXl04D4Aw&bvm=bv.44442042,d.dGI&psig=AFQjCNGMhqaM1n0RbUTg6WP7blwbbvjUPg&ust=1364882267848704http://www.google.co.kr/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=FkWiA-aB9a-maM&tbnid=eN2fAWjK6g5kGM:&ved=0CAUQjRw&url=http://www.jenabioscience.com/images/1b94dca880/Newsletter_Fluoro-ddNTPs_RT-PCR_Oct08.html&ei=9idZUZyFKYePiAeOsoCoDA&bvm=bv.44442042,d.aGc&psig=AFQjCNFUr-xqj9hs0N6w_678tshDOfRlGw&ust=1364883752401211

Capillary gel electrophoresis is used to read the sequence

-Capillary gel is a long, thin tube of polyacrylamide. the mixture of chain-terminated molecules is placed at one end of the capillary and an electric current applied

1) Each phosphodiester bond in a DNA moleculecarries a single negative electric charge, sothe overall negative charge of apolynucleotide is directly proportional to itslength

2) The chain-terminated molecules thereforemigrate through the capillary gel atdifferent rates, the shortest ones mostquickly and longer one the slowest.

3) Each of the four dideoxynucleotides emits afluorescence signal of a differentwavelength

4) A fluorescence detector is therefore usedto identify which of the fourdideoxynucleotides is present in each band

Dye-terminator sequencing

Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths. Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis (see figure to the left). This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of

sequencing projects.

http://en.wikipedia.org/wiki/Wavelengthhttp://en.wikipedia.org/wiki/Chromatogramhttp://en.wikipedia.org/wiki/Capillary_electrophoresis

Genome sequence project

1) The first step n a DNA sequencing project isto prepare a collection of short DNA fragmentby sonication (high-frequency sound waves tomake random cuts in the molecules)

2) Each fragment is cloned into E. Coli (A), and aDNA molecule that is able to replicate inside a cell of the bacterium called “Vector”.

3) The vector is usually a modified version of anaturally occurring, self-replicating DNA molecule called a plasmid.

4) Each bacteria(colony) contains many copies ofvector containing multiple copies of a single DNA fragment.

5) Primers are added to start polymerase sequencereaction

Immediately we can understand why big sequencing experiments carried out by robots, and computer programs used to find the overlaps and build up the contiguous blocks of sequence or contigs.

A maximum of about 750 bp of sequence can be obtained in a single experiment. Automated sequencers with multiple capillaries working can read up to 96 different sequences in a 2-hr, which can sequence 864,000 bp of information can be generated per day.- 23 paired chromosome three billion DNA base pairs ??

http://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.html

The $3-billion project was formally founded in 1990 by the United States Department of Energy and the U.S. National Institutes of Health, and was expected to take 15 years. In addition to the United States, the international consortium comprised geneticists in the United Kingdom, France, Germany, Japan, China, and India.

Due to widespread international cooperation and advances in the field of genomics (especially in sequence analysis), as well as major advances in computing technology, a 'rough draft' of the genome was finished in 2000 (announced jointly by then US president Bill Clinton and the British Prime Minister Tony Blair on June 26, 2000).[7] This first available rough draft assembly of the genome was completed by the UCSC Genome Bioinformatics Group, primarily led by then graduate student Jim Kent. Ongoing sequencing led to the announcement of the essentially complete genome in April 2003, 2 years earlier than planned.[8] In May 2006, another milestone was passed on the way to completion of the project, when the sequence of the last chromosome was published in the journal Nature.[9]

http://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://www.dnalc.org/view/15073-Completion-of-a-draft-of-the-human-genome-Bill-Clinton.htmlhttp://en.wikipedia.org/wiki/Research_fundinghttp://en.wikipedia.org/wiki/Research_fundinghttp://en.wikipedia.org/wiki/Research_fundinghttp://en.wikipedia.org/wiki/United_States_Department_of_Energyhttp://en.wikipedia.org/wiki/U.S._National_Institutes_of_Healthhttp://en.wikipedia.org/wiki/U.S._National_Institutes_of_Healthhttp://en.wikipedia.org/wiki/United_Stateshttp://en.wikipedia.org/wiki/Consortiumhttp://en.wikipedia.org/wiki/Geneticisthttp://en.wikipedia.org/wiki/United_Kingdomhttp://en.wikipedia.org/wiki/Francehttp://en.wikipedia.org/wiki/Germanyhttp://en.wikipedia.org/wiki/Japanhttp://en.wikipedia.org/wiki/Chinahttp://en.wikipedia.org/wiki/Indiahttp://en.wikipedia.org/wiki/Genomicshttp://en.wikipedia.org/wiki/Sequence_analysishttp://en.wikipedia.org/wiki/Bill_Clintonhttp://en.wikipedia.org/wiki/United_Kingdomhttp://en.wikipedia.org/wiki/Prime_Minister_of_the_United_Kingdomhttp://en.wikipedia.org/wiki/Tony_Blairhttp://en.wikipedia.org/wiki/Genome_Projecthttp://en.wikipedia.org/wiki/UCSChttp://en.wikipedia.org/wiki/Jim_Kenthttp://en.wikipedia.org/wiki/Sequencinghttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Chromosome_1_(human)http://en.wikipedia.org/wiki/Nature_(journal)http://upload.wikimedia.org/wikipedia/en/6/60/DNA_Sequencing_gDNA_libraries.jpg

Frederick Sanger, (born 13 August 1918) is an English biochemist and twice a Nobel laureate in chemistry. He is the fourth (and only living) person to have been awarded two Nobel Prizes.

http://en.wikipedia.org/wiki/Englandhttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Nobel_laureatehttp://en.wikipedia.org/wiki/Chemistryhttp://en.wikipedia.org/wiki/Nobel_Prize

Maxim and Gilbert chemical sequencing method is based on the ability of different chemical reagents like hydrazine, dimethyl sulfate(DMS), or formic acid to specifically modify bases within the DNA molecule. This modification is done under mild condition that lead to an average of one specific methylated base per DNA strand. Next we use piperidine that does two things- removes the methylated base & then it breaks the DNA beck bone at the site of the lost base that is called the apurinic or apyrimidimic site G: DMS G+A: Formic acid T+C: Hydrazine C: in the presence of NaCl, only C reacts with hydrazine

Human Genome Project began in 1985, TiGR- Facilities (Crag Venter)

Some of the first commercially available Next Generation technologies were developed by 454 Life Sciences and Solexa technology, later purchased by Illumina. Both methods involve random shearing of genomic DNA, followed by linking to beads or a specialised slide.

The 454 Life Sciences system is based on the ‘pyrosequencing’ method, which allows shotgun sequencing without cloning any of the DNA

During a DNA synthesis reaction, a Phosphate group is released when two nucleotides are bound. Pyrosequencing measures the amount of phosphate released as each dNTP is added to the reaction and incorporated into the sequence

Solexa technology [Fig.3B] also uses DNA synthesis reactions but measuring fluorescence rather than pyrophosphate reselase. Fluorescently labeled chain-terminating nucleotides are incorporated into the sequence and measured by a detector.

4주 DNA-replicatin 2 (PCR&Sequencing)4슬라이드 번호 1슬라이드 번호 2Human Genome Project began in 1985, TiGR- Facilities (Crag Venter) Some of the first commercially available Next Generation technologies were developed by 454 Life Sciences and Solexa technology, later purchased by Illumina. Both methods involve random shearing of genomic DNA, followed by linking to�beads or a specialised slide. ��The 454 Life Sciences system is based on the ‘pyrosequencing’ method, which allows shotgun sequencing without cloning any of the DNA