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Images from the Text are protected by Copyright (c) 2008 by W. H. Freeman and Company, and by the licensors of W. H. Freeman and Company. Living Graphs software (c) 2008 Sumanas, Inc. ALL RIGHTS RESERVED.
Commentary by the instructor is protected by Copyright (c) 2011. ALL RIGHTS RESERVED.
BCH 5045
Graduate Survey of Biochemistry
Instructor: Charles Guy Producer: Ron Thomas
Director: Marsha Durosier
Lecture 19 Slide sets available at:
http://hort.ifas.ufl.edu/teach/guyweb/bch5045/index.html
Images from the Text are protected by Copyright (c) 2008 by W. H. Freeman and Company, and by the licensors of W. H. Freeman and Company. Living Graphs software (c) 2008 Sumanas, Inc. ALL RIGHTS RESERVED.
Commentary by the instructor is protected by Copyright (c) 2011. ALL RIGHTS RESERVED.
• LEHNINGER • PRINCIPLES OF BIOCHEMISTRY
• Fifth Edition
David L. Nelson and Michael M. Cox
© 2008 W. H. Freeman and Company
CHAPTER 9 DNA-Based Information Technologies
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Commentary by the instructor is protected by Copyright (c) 2011. ALL RIGHTS RESERVED.
Here the Sanger dideoxy method of DNA sequencing is illustrated. The Key elements are primers, dideoxy nucleotide triphosphates, regular deoxynucleotide triphosphates with 32P labeled dNTPs or more common with sequencing machines attached to fluorescent tags, template DNA and a DNA polymerase. Can you tell how this works?
Images from the Text are protected by Copyright (c) 2008 by W. H. Freeman and Company, and by the licensors of W. H. Freeman and Company. Living Graphs software (c) 2008 Sumanas, Inc. ALL RIGHTS RESERVED.
Commentary by the instructor is protected by Copyright (c) 2011. ALL RIGHTS RESERVED.
Images from the Text are protected by Copyright (c) 2008 by W. H. Freeman and Company, and by the licensors of W. H. Freeman and Company. Living Graphs software (c) 2008 Sumanas, Inc. ALL RIGHTS RESERVED.
Commentary by the instructor is protected by Copyright (c) 2011. ALL RIGHTS RESERVED.
Images from the Text are protected by Copyright (c) 2008 by W. H. Freeman and Company, and by the licensors of W. H. Freeman and Company. Living Graphs software (c) 2008 Sumanas, Inc. ALL RIGHTS RESERVED.
Commentary by the instructor is protected by Copyright (c) 2011. ALL RIGHTS RESERVED.
A primer is hybridized to a single stranded PCR amplified DNA template and mixed with DNA polymerase, ATP sulfurylase, luciferase and apyrase, and adenosine 5´ phosphosulfate and luciferin. One of the four dNTPs is added. The DNA polymerase catalyzes the incorporation of the dNTP to the 3 ´ end of new strand according to the template strand. The addition of the dNTP to the 3 ´ end of the new DNA strand releases pyrophosphate (PPi). ATP sulfurylase converts PPi to ATP which is used to drive the luciferase mediated conversion of luciferin to oxyluciferin which produces light in proportion to the amount of ATP made which is proportional to the amount of the nucleotide added to the growing DNA strand. The light is detected by a CCD camera and appears as a peak on the pyrogram. The height of the peak is proportional to the number of the particular nucleotide incorporated into the DNA strand. Apyrase which degrades nucleotides is added which will act to block the light production. Addition of the next dNTP starts the process all over again.
Essence of Pyrosequencing
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Commentary by the instructor is protected by Copyright (c) 2011. ALL RIGHTS RESERVED.
PYROSEQUENCING is a unique method for short-read DNA sequencing and single nucleotide sequence variation analysis. Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle developed by Mostafa Ronaghi and Pål Nyrén (Analytical Biochemistry 1996 and Science 1998). Pyrosequencing AB initially began to commercialize the technology for sequencing of short stretches of DNA. Pyrosequencing AB was renamed to Biotage in 2003 and the technology was further licensed to 454 Life Sciences. 454 developed an array-based pyrosequencing platform for large-scale DNA sequencing. Most notably, are its applications for genome sequencing and metagenomics. The latest platform of pyrosequencing the GS FLX from 454 Life Sciences owned by Roche can generate 100 million nucleotide dataset in a 7 hour run. It is expected that throughput could increase by 5-10 fold with the next version of the platform. Thus, each run could cost under $10,000 and provide the de novo sequencing of mammalian genomes in about the $1,000,000 range.
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The method is based on a chemical light-producing enzymatic reaction, which occurs when a molecular recognition event occurs. The sequencing of a single strand of DNA by synthesizing the complementary strand is the basis of the method. Each time a nucleotide, A, C, G or T is incorporated into the growing chain a cascade of enzymatic reactions is triggered which causes a light signal.
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Let’s look at the sequencing by synthesis a little more A ssDNA template is hybridized to a sequencing primer and incubated with
DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin.
1. The addition of a deoxynucleotide triphosphate (dNTP) initiates the
second step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically as in standard DNA synthesis.
2. ATP sulfurylase converts PPi to ATP in the presence of adenosine 5´ phosphosulfate.
3. The ATP the allows a luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a CCD camera and analyzed. Each light signal is proportional to the number of nucleotides incorporated.
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4. Unincorporated nucleotides and ATP are degraded by apyrase, and the reaction can restart with another nucleotide.
A limitation of the method is that individual reads of DNA sequence are 300-500 nucleotides in length. By 2007, pyrosequencing is most commonly used for resequencing or sequencing of genomes for which the sequence of a close relative is already known, and the reads are becoming longer.
Its ease, sequence validation and flexibility makes it well suited for applied genomics research including molecular applications for disease diagnosis, clinical prognosis and drug testing. A typical run time is 10 minutes for 96 samples and approximately 30 to 45 minutes for sequence analysis applications that routinely provide 30 to 50 bases of sequence information. No gels, radioactivity or dyes are needed. Pyrosequencing is unprecedented in that it is uniquely suited and highly powerful for certain applications. One example would be quantitative analysis of Prader-Willi syndromes by pyrosequencing.
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Prader-Willi / Angelmann developmental syndromes usually arise from a deletion of a differentially methylated DNA region on the human chromosome 15q11-13. Different patterns of DNA methylation are detected depending on which pair of the chromosome is deleted. A maternally deleted chromosome leads to hypomethylation, whereas hypermethylation is seen when the paternal chromosome is deleted. Pyrosequencing was able to provide a simple and fast technique to detect the two syndromes. Based on Pyrosequencing’s quantitative properties, assessment of chromosomal methylation patterns is more accurate, sensitive and reproducible than by any other technique, with results available 30 minutes after PCR.
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Ronaghim M. 2001. Pyrosequencing Sheds Light on DNA Sequencing. Genome Res. Vol. 11: 3-11.
Ahmadian, A, Ehn, M, Hober S (2006) Pyrosequencing: History, biochemistry and future Clinica Chimica Acta 363: 1-2, 83-94
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Sundquist A, Ronaghi M, Tang H, Pevzner P, Batzoglou S. (2007) Whole-genome sequencing and assembly with high-throughput, short-read technologies. PLoS ONE. 2007 May 30;2(5):e484.
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Now the Next Generation Sequencing
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This slide not in the lecture video The next generation of DNA sequencing (perhaps could be called nextgen sequencing 3.0) has already begun. In 2009, Eid et al published in Science a paper describing a new sequencing strategy that they refer to as “Real-Time DNA Sequencing from Single Polymerase Molecules” which is also the title of their paper. They used “DNA polymerase as a real-time sequencing engine” where the processive nature of DNA polymerization can be directly observed with individual base-pair resolution taking advantage of the speed, fidelity, and processivity of the polymerase. They employed a nanophotonic structure, the zero-mode waveguide. This allowed for a reduction in the volume of the reaction mixture by three orders of magnitude beyond the resolution of a confocal fluorescence microscope. This makes possible single-fluorophore detection at labeled-dNTP concentrations of 0.1 up to 10 μM. Binding of the correct base-paired phospholinked dNTPs (cognate) in the active site of the polymerase gives rise to a fluorescence pulse by the polymerase retaining the cognate nucleotide with its color-coded fluorophore in the detection region of the zero-mode waveguide. The florescence duration depends on the rate of catalysis but ends with the cleavage of the dye-linker-pyrophosphate group that quickly diffuses away from the zero-mode waveguide detection region. Insertion errors were observed when a cognate nucleotide dissociated from the active site before phosphodiester bond formation occurred giving a duplication of a florescence pulse.
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Advantages of the Eid et al sequencing system include: Listen to podcast
• Longer sequence reads
• Less DNA needed for sequencing
• Vastly reduced reagents needed for sequencing reaction
• Speed of sequencing at the rate of polymerization of DNA polymerase
• Occupies small space that can allow massively parallel sequencing reaction
platform
• Fluorophore linked to the dNTP at the terminal phosphate moiety
(phospholinked), thus DNA polymerase catalyzed phosphodiester bond formation
results in release of the fluorophore from the incorporated nucleotide yielding a
native DNA strand that doesn’t compromise or interfere with the function of the
polymerase to continue its catalytic cycle
This slide not in the lecture video
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Fig. 1. Principle of single-molecule, real-time DNA sequencing.
J Eid et al. Science 2009;323:133-138
Published by AAAS
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Fig. 2. Real-time detection of single-molecule DNA polymerase activity.
J Eid et al. Science 2009;323:133-138
Published by AAAS
Images from the Text are protected by Copyright (c) 2008 by W. H. Freeman and Company, and by the licensors of W. H. Freeman and Company. Living Graphs software (c) 2008 Sumanas, Inc. ALL RIGHTS RESERVED.
Commentary by the instructor is protected by Copyright (c) 2011. ALL RIGHTS RESERVED.
Images from the Text are protected by Copyright (c) 2008 by W. H. Freeman and Company, and by the licensors of W. H. Freeman and Company. Living Graphs software (c) 2008 Sumanas, Inc. ALL RIGHTS RESERVED.
Commentary by the instructor is protected by Copyright (c) 2011. ALL RIGHTS RESERVED.
Human Genome Sequence Project
Synteny, what is it?
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Genome Databases and Information