BCH 5045 Graduate Survey of Biochemistryhort.ifas.ufl.edu/faculty/guy/bch5045/Lecture Files/Lecture 19.pdf · CHAPTER 9 . DNA-Based Information Technologies . ... The chemical synthesis

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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

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• 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



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?

Presenter

Presentation Notes

FIGURE 8-33c DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (c) The DNA to be sequenced is used as the template strand, and a short primer, radioactively or fluorescently labeled, is annealed to it. By addition of small amounts of a single ddNTP, for example ddCTP, to an otherwise normal reaction system, the synthesized strands will be prematurely terminated at some locations where dC normally occurs. Given the excess of dCTP over ddCTP, the chance that the analog will be incorporated whenever a dC is to be added is small. However, ddCTP is present in sufficient amounts to ensure that each new strand has a high probability of acquiring at least one ddC at some point during synthesis. The result is a solution containing a mixture of labeled fragments, each ending with a C residue. Each C residue in the sequence generates a set of fragments of a particular length, such that the different-sized fragments, separated by electrophoresis, reveal the location of C residues. This procedure is repeated separately for each of the four ddNTPs, and the sequence can be read directly from an autoradiogram of the gel. Because shorter DNA fragments migrate faster, the fragments near the bottom of the gel represent the nucleotide positions closest to the primer (the 5′ end), and the sequence is read (in the 5′→3′ direction) from bottom to top. Note that the sequence obtained is that of the strand complementary to the strand being analyzed.



Presenter

Presentation Notes

FIGURE 8-34 Strategy for automating DNA sequencing reactions. Each dideoxynucleotide used in the Sanger method can be linked to a fluorescent molecule that gives all the fragments terminating in that nucleotide a particular color. All four labeled ddNTPs are added to a single tube. The resulting colored DNA fragments are then separated by size in a single electrophoretic gel contained in a capillary tube (a refinement of gel electrophoresis that allows for faster separations). All fragments of a given length migrate through the capillary gel in a single peak, and the color associated with each peak is detected using a laser beam. The DNA sequence is read by determining the sequence of colors in the peaks as they pass the detector. This information is fed directly to a computer, which determines the sequence.



Presenter

Presentation Notes

FIGURE 8-35 Chemical synthesis of DNA by the phosphoramidite method. Automated DNA synthesis is conceptually similar to the synthesis of polypeptides on a solid support. The oligonucleotide is built up on the solid support (silica), one nucleotide at a time, in a repeated series of chemical reactions with suitably protected nucleotide precursors. 1 The first nucleoside (which will be the 3′ end) is attached to the silica support at the 3′ hydroxyl (through a linking group, R) and is protected at the 5′ hydroxyl with an acid-labile dimethoxytrityl group (DMT). The reactive groups on all bases are also chemically protected. 2 The protecting DMT group is removed by washing the column with acid (the DMT group is colored, so this reaction can be followed spectrophotometrically). 3 The next nucleotide has a reactive phosphoramidite at its 3′ position: a trivalent phosphite (as opposed to the more oxidized pentavalent phosphate normally present in nucleic acids) with one linked oxygen replaced by an amino group or substituted amine. In the common variant shown, one of the phosphoramidite oxygens is bonded to the deoxyribose, the other is protected by a cyanoethyl group, and the third position is occupied by a readily displaced diisopropylamino group. Reaction with the immobilized nucleotide forms a 5′,3′ linkage, and the diisopropylamino group is eliminated. In step 4, the phosphite linkage is oxidized with iodine to produce a phosphotriester linkage. Reactions 2 through 4 are repeated until all nucleotides are added. At each step, excess nucleotide is removed before addition of the next nucleotide. In steps 5 and 6 the remaining protecting groups on the bases and the phosphates are removed, and in 7 the oligonucleotide is separated from the solid support and purified. The chemical synthesis of RNA is somewhat more complicated because of the need to protect the 2′ hydroxyl of ribose without adversely affecting the reactivity of the 3′ hydroxyl.



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



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.



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.



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.



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.



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.



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



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.



Now the Next Generation Sequencing

The viewing of a commercial company’s website does not constitute an endorsement by the instructor, College or University of the company’s products.

http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems.html�

http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html�



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.



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

http://www.sciencemag.org/content/323/5910/133/suppl/DC2�



Fig. 1. Principle of single-molecule, real-time DNA sequencing.

J Eid et al. Science 2009;323:133-138

Published by AAAS

Presenter

Presentation Notes

Principle of single-molecule, real-time DNA sequencing. (A) Experimental geometry. A single molecule of DNA template-bound Φ29 DNA polymerase is immobilized at the bottom of a ZMW, which is illuminated from below by laser light. The ZMW nanostructure provides excitation confinement in the zeptoliter (10–21 liter) regime, enabling detection of individual phospholinked nucleotide substrates against the bulk solution background as they are incorporated into the DNA strand by the polymerase. (B) Schematic event sequence of the phospholinked dNTP incorporation cycle, with a corresponding expected time trace of detected fluorescence intensity from the ZMW. (1) A phospholinked nucleotide forms a cognate association with the template in the polymerase active site, (2) causing an elevation of the fluorescence output on the corresponding color channel. (3) Phosphodiester bond formation liberates the dye-linker-pyrophosphate product, which diffuses out of the ZMW, thus ending the fluorescence pulse. (4) The polymerase translocates to the next position, and (5) the next cognate nucleotide binds the active site beginning the subsequent pulse.



Fig. 2. Real-time detection of single-molecule DNA polymerase activity.

J Eid et al. Science 2009;323:133-138

Published by AAAS

Presenter

Presentation Notes

Real-time detection of single-molecule DNA polymerase activity. (A) DNA template design for two-base sequence pattern detection. The sequence of a linear, single-stranded DNA template was designed to yield incorporation of alternating blocks of two phospholinked nucleotides (A555-dCTP and A647-dGTP), interspersed with the other two, unmodified dNTPs. (B) Time-resolved fluorescence intensity spectrum from a ZMW. Data from a 15 × 5 pixel area from each movie frame were spatially collapsed to a 15-pixel spectrum, which is shown as a function of time. The expected fluorescence emission profiles for the two labeled nucleotides are shown at the right. The arrow denotes addition of the catalytic metal ion that initiated the polymerization reaction. The complete data set from which the time trace was extracted, containing 3000 ZMWs measured simultaneously, is shown in movie S1. (C) Corresponding fluorescence time trace after spectral processing. Two regions are magnified and annotated with the expected nucleotide incorporation sequence. Pulse heights show level setting with a coefficient of variation of 27% and a maximal excursion of 61%.





Human Genome Sequence Project

Synteny, what is it?

Presenter

Presentation Notes

FIGURE 9-18 Genomic sequencing timeline. Discussions in the mid-1980s led to initiation of the Human Genome Project in 1989. Preparatory work, including extensive mapping to provide genome landmarks, occupied much of the 1990s. Separate projects were launched to sequence the genomes of other organisms important to research. The sequencing efforts completed to date include many bacterial species (such as Haemophilus influenzae), yeast (S. cerevisiae), nematode worms (e.g., C. elegans), insects (D. melanogaster and Apis mellifera), plants (A. thaliana and Oryza sativa L.), rodents (Mus musculus and Rattus norvegicus), primates (Homo sapiens and Pan troglodytes), and some nasty human pathogens (e.g., Trichomonas vaginalis). Each genome project has a website that serves as a central repository for the latest data. FIGURE 9-19 Snapshot of the human genome. The chart shows the proportions of our genome made up of various types of sequences. FIGURE 9-20 Synteny in the mouse and human genomes. Large segments of the mouse and human genomes have closely related genes aligned in the same order on chromosomes, a relationship called synteny. This diagram shows segments of human chromosome 9 and mouse chromosome 2. The genes in these segments exhibit a very high degree of homology as well as the same gene order. The different lettering schemes for the gene names reflect different naming conventions in the two organisms.



Genome Databases and Information

http://www.ncbi.nlm.nih.gov/�

http://www.ncbi.nlm.nih.gov/sites/genome�

http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi�

http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj�

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BCH 5045 Graduate Survey of Biochemistryhort.ifas.ufl.edu/faculty/guy/bch5045/Lecture Files/Lecture 19.pdf · CHAPTER 9 . DNA-Based Information Technologies . ... The chemical synthesis