ABT 605 MOLECULAR DIAGNOSTICSTERM PAPER DNA SEQUENCING-PYRO SEQUENCINGSubmitted byP.S.VINODI M.V.Sc- Animal BiotechnologyMadras Veterinary college

DNA SEQUENCING-

PYRO SEQUENCING-A brief compilation of literature

DEOXYRIBONUCLEIC ACID SEQUENCING A procedure to determine the order of nucleotides /the order of the four bases viz adenine, guanine, cytosine, and thymine within a Deoxyribo nucleic acid molecule.

NEED FOR SEQUENCINGSome important applications of DNA sequencing are :Basic biological researchTo analyse any protein structure and function we must have the knowledge of its primary structure i.e. its DNA sequence.DNA sequence is use to understand the function of a specific sequence and the sequence responsible for any disease.Comparative DNA sequence study are used to detect any mutation.Kinship study.DNA fingerprinting.By knowing the whole genome sequence, Human genome project got completed.

DETERMINING THE SEQUENCE OF DNAChemical method- Maxim and Gilbert methodChain termination method- F .Sanger Sangers method

A modification Shotgun sequencingII Generation SequencingDNA Polymerase Dependent Strategies - Single Nucleotide addition Sequencing by synthesis -Pyrosequencing

Next Generation Sequencing454 Pyro sequencingCyclic reversible termination - Illumina/Solexa sequencing platform DNA ligase dependent strategy of sequencing by ligation-The Applied Biosystems Sequencing by Oligonucleotide Ligation and Detection (SOLiD) system.

Pyro sequencing A real time non electrophoretic ,non fluorescent DNA sequencing method based on Sequencing by synthesis principle that measures/relies on the release of organic pyrophosphate (PPi) during DNA polymerisation reaction which is proportionately converted into visible light by series of enzymatic reactions.Brief HistoryPyrosequencing conceived by Pal Nyren in 1986 in the laboratory of Sir John Walker- MRC laboratory of Molecular Biology England 1987 - Pal Nyren demonstrated monitoring of DNA polymerase activity using bioluminescence.1996-Pal Nyrens group reported that natural nucleotides could be used to obtain efficient incorporation during sequencing by synthesis protocolPyrosequencing has emerged as an alternative method of sequencing. Although it has read-length limitations compared with di-deoxy sequencing, it is a fast method with real-time read-out that is highly suitable for sequencing short stretches of DNA (Gharizadeh et al., 2007).

PrincipleUses the basic properties of DNA bases and polymerisation vizProperty of the nucleotides to pair specifically to their complementary nucleotides and Release of pyrophosphate when a nucleotide base pairs with its complementary nucleotide in a DNA polymerase catalyzed reactionIn the polymerase reaction, the 3' hydroxyl group on the end of the growing DNA strand attacks the a-phosphate of a 2'-deoxynucleoside triphosphate, expelling inorganic pyrophosphate.

Principle (continued)The released pyrophosphate is subsequently converted to ATP by ATP sulfurylase using Adenosine 8 phosphosulfate.The ATP produce provides energy for oxidation of luciferin The oxidation of luciferin produces light proportional to the number of ATP formed.One pico mol of DNA in a Pyrosequencing reaction yields 6x1011 ATP molecules which in turn can generate more than 6x109 photons at a wavelength of 560 nm.The light generated is captured by a photodiode / photomultiplier tube /Charged Coupled Device camera and recorded in the form of peaks known as PyrogramWhen a nucleotide is not incorporated into the reaction, no pyrophosphate is released and the unused nucleotide is removed from the system by degradation through apyrase.A cycle takes 3-4 seconds at room temperature

Pyrosequencing techniques in detailTo avoid interference by unincorporated nucleotides and primers used in PCR the template is purified prior to sequencing.Strategies1.Solid phase Pyrosequencing or three enzyme procedureOnly three enzymes are utilised in this procedure i.e DNA polymerase,

Sulfurylase and luciferaseTo remove the non incorporated deoxynucleotides and ATP resulting from

sulfurylase action a template washing step is required between nucleotide addition.Biotinylated PCR products are immobilized by capture onto magnetic beadsAfter PCR and sedimentation the DNA obtained is washed and alkali treated to

obtain single stranded DNA.Both immobolized biotinylated and non biotinylated strands in is solution are

used as template

1.Solid phase Pyrosequencing or three enzyme procedure

2.Liquid phase Pyrosequencing or four enzyme mixture Four enzymes are utilised in this procedure i.e DNA polymerase, Sulfurylase luciferase and apyraseApyrase eliminates the need for immobilization and intermediate washing stepApyrase degrades unused nucleotides not incorporated in the reactionPerformed in a closed system in a single well.The processing is speeded up and a 96 well plate is read in approximately 20 minutes

2.Liquid phase Pyrosequencing or four enzyme mixture

Enzymes and reagentsKlenow fragment of E.coli DNA pol I ( a relatively slow polymerase)ATP sulfurylase (a recombinant version obtained from yeast S.cerevisiaeLuciferase enzyme (from American firefly Photinus pyralis-can produce 0.88 photons per luciferin molecule in green yellow region 550-590nm)Apyrase (pimpernal apyrase from Solanum tuberosum)Adenosine 5 phosphosulfate (APS)D-Luciferin (Substrate for luciferase)dNTPs ( dCTP,dGTP,dTTP,dATPS*)Buffers

* To avoid nonspecific signals by dATP, luciferase reaction and also to enable reaction in homogenous phase and real time

The ReactionsIn each cycle the reaction is separately exposed to each of the four nucleotides i.e. dNTPsWhen correct nucleotide is incorporated(DNA)n +dNTPs ( DNA)n+1 +

+ adenosine 5 phosphosulfate(APS) ATP + SO42- Luciferase + d-luciferin +ATP luciferase-luciferin- AMP+PpiLuciferase-luciferin AMP+O2 -luciferase+ oxyluciferin +AMP+CO2 + (Light)

PPiPPiDNA PolymeraseSulfurylaseUnincorporated nucleotides and ATP between addition of bases

ATP AMP+2Pi

dNTP deoxy nucleotide monophosphate +2PiApyraseluciferaseApyrase

Schematic representation of PyrosequencingAm. J. Biochem. & Biotech., 8 (1): 14-20, 2012Md. Fakruddin and Abhijit Chowdhury

MethodThe DNA from samples are amplified using PCR with primers Prepare PCR plate for PyrosequencingPrepare Pyrosequencing plate Set up workstation for prepping PCR product/bead mix tray and Pyrosequencing primer tray Enter assay detailsFill the cartridge with reagents and place it in the machine(PSQHS96A)Test run with the reagent filled cartridgeRun new SNPAnalyse results

Pyrosequencing Results:

Application of pyrosequencing: Pyrosequencing is well suited for de novo sequencing and resequencing (Ronaghi, 2001). Pyrosequencing method is broadly being used in many applications such as Single Nucleotide Polymorphism (SNP) genotyping (Ahmadian et al., 2000a; Nordstrom et al., 2000; Milan et al., 2000), identification of bacteria (Gharizadeh, 2003; Grahn et al., 2003; Jonasson et al., 2002), identification of Fungal types (Gharizadeh et al., 2005; Trama et al., 2005) Viral typing (Gharizadeh et al., 2001; 2003; 2005; Adelson et al., 2005). The method has demonstrated the ability to determine difficult secondary structures (Ronaghi, 2001)Perform mutation detection (Ahmadian et al., 2000b; Garcia et al., 2000), DNA methylation analysis (Neve et al., 2002; Uhlmann et al., 2002),

multiplex sequencing (Gharizadeh et al., 2003a; 2006) (Gharizadeh et al., 2003b;

Gharizadeh et al., 2006),tag sequencing of cDNA library (Nordstrom et al., 2001) and clone checking

(Nourizad et al., 2003). Another highly significant application is whole genome sequencing (Margulies

et al., 2005). the technology can be applied to study Single-nucleotide polymorphisms (SNP), insertion/deletions (indels), short tandem repeats, pooled allele frequencies, human leukocyte antigen (HLA) typing, gene copy number, allelic imbalance in RNA, methylation status, and short sequencing stretches,

Application of pyrosequencing: Continued

. Application of pyrosequencing: Continued

As short stretches of sequence are synthesized during the assay, novel polymorphisms close to the polymorphism in question have also been identified using this technique, where they may be missed or cause inaccurate genotype calls using other methods. The assay is applicable to almost any source of DNA or RNA (e.g., blood, saliva, cell line, plasma, serum, tissue, formalin fixed and/or paraffin-embedded samples, and whole genome-amplified DNA).In addition, the use of a universal biotinylated primer and multiplex analysis of

up to three different amplicons can be performed reducing genotypingcost and time of throughput. No other system provides this range, throughput,and cost advantage.

Advantages:AccurateParallel processingEasily automatedEliminates the need for labeled primers and nucleotidesNo need for gel electrophoresis

An important factor in pyrosequencing is primer design for PCR and sequencing. Sequencing primers should be checked for self-looping, primer-dimer (primer-primer hybridizations) and cross-hybridization (when more than one sequencing primer is used). (Gharizadeh et al., 2007).An inherent problem with the described method is de novo sequencing of polymorphic regions in heterozygous DNA material (Ronaghi, 2001).Another inherent problem is the difficulty in determining the number of incorporated nucleotides in homopolymeric regions, due to the nonlinear light response following incorporation of more than 5-6 identical nucleotides (Ronaghi, 2001).

454 PyrosequencingA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics. To start, the DNA is sheared into 300-800 bp fragments, and the ends are polished by removing any unpaired bases at the ends.Adapters are added to each end. The DNA is made single stranded at this point.One adapter contains biotin, which binds to a streptavidin-coated bead. The ratio of beads to DNA molecules is controlled so that most beads get only a single DNA attached to them.Oil is added to the beads and an emulsion is created. PCR is then performed, with each aqueous droplet forming its own micro-reactor. Each bead ends up coated with about a million identical copies of the original DNA.

454 Pyrosequencing Contd..

After the emulsion PCR has been performed, the oil is removed, and the beads are put into a picotiter plate. Each well is just big enough to hold a single bead.The pyrosequencing enzymes are attached to much smaller beads, which are then added to each well.The plate is then repeatedly washed with the each of the four dNTPs, plus other necessary reagents, in a repeating cycle.The plate is coupled to a fiber optic chip. A CCD camera records the light flashes from each well.

Comparison of next-generation sequencing methods[52][53]Quail MA, Smith M, Coupland P, Otto TD, Harris SR, Connor TR et al. (1 January 2012). "A tale of three next generation sequencing platforms: comparison of Ion torrent, pacific biosciences and illumina MiSeq sequencers". BMC Genomics 13 (1): 341. doi:10.1186/1471-2164-13-341. PMC3431227. PMID22827831. Jump up ^ Liu L, Li Y, Li S, Hu N, He Y, Pong R et al. (1 January 2012). "Comparison of Next-Generation Sequencing Systems". Journal of Biomedicine and Biotechnology (Hindawi Publishing Corporation) 2012: 111. doi:10.1155/2012/251364. PMID22829749.

the NGS platforms read many more DNAs in parallel but have shorter read lengths. For example, 454s GS FLX machine reads 400,000 DNAs that are each about 250 bases in length; the 454 machine sequences are about ten times cheaper than traditional Sanger technologyIlluminas Genome Analyzer and ABIs SOliD platform can read tens of millions of DNAs up to about 3550 bases in length.

These three NGS platforms also differ in cost per base: Illumina and ABI are 100 times cheaper,- Steven Jones, head of bioinformatics

at the Genome Sciences Centre in Vancouver, Canada.Next-Generation Sequencing: The Race Is OnCell 132, March 7, 2008 2008 Elsevier Inc. 721

Reference

Comparative Genomic Basic and Applied Research James R.Brown 2008Methods in Molecular Biology, vol. 373: Pyrosequencing Protocols

Edited by: S. Marsh Humana Press Inc., Totowa, NJGenomics fundamentals and applications Supratim chaudari &David B Carlson 2009Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Research. 2001: 11: 3-11Nordstrom T., Ronaghi M., Forsberg L., de Faire U., Morgenstern R. and Nyren P. Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing.Biotechnol. Applied Biochem. 2000: 31: 107- 112.Nordstrom T., Gharizadeh B., Pourmand N., Nyren P. and Ronaghi M. Method enabling fast partial sequencing of cDNA clones. Anal. Biochem. 2001: 292: 266-271.Nordstrom T., Ronaghi M., Forsberg L., de Faire U., Morgenstern R. and Nyren P. Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing. Biotechnol. Appl. Biochem. 2000: 31: 107 112.

Ahmadian A., Gharizadeh B., Gustafsson A.C., Sterky F., Nyren P., Uhlen M. andLundeberg J. Single-nucleotide polymorphism analysis by pyrosequencing. Anal. Biochem. 2000a: 280: 103110. Ahmadian A., Lundeberg J., Nyren P., Uhlen M. and Ronaghi M. Analysis of the p53 tumor suppressor gene by pyrosequencing. Biotechniques, 2000b: 28: 140147.Gharizadeh B., Ghaderi M. and Nyren P. Pyrosequencing technology for short DNA sequencing and whole genome sequencing. Technology. 2007: 47: 129-132. Gharizadeh B., Nordstrom T., Ahmadian A., Ronaghi M. and Nyren P. Long-read pyrosequencing using pure 2- deoxyadenosine-5-O-(1-thiotriphosphate)Sp-isomer. Anal. Biochem. 2002: 301: 82-90.Gharizadeh B., Eriksson J., Nourizad N., Nordstrom T. and Nyren P. Improvement in pyrosequencing technology by employing sequenase polymerase. Anal. Biochem. 2004: 330: 272-280.Adelson M.E., Feola M., Trama J., Tilton R.C. and Mordechai E. Simultaneous detection of herpes simplex virus types 1 and 2 by realtime PCR and pyrosequencing. J. Clin. Virol. 2005: 33: 25-34.Jonasson J., Olofsson M. and Monstein H.J. Classification, identification and subtyping of bacteria based on pyrosequencing and signature matching of 16S rDNA fragments. APMIS. 2002: 110: 263272.

Trama J.P., Mordechai E. and Adelson M.E. Detection of Aspergillus fumigatus and a mutation that confers reduced susceptibility to itraconazole and posaconazole by real-time PCR and pyrosequencing. J. Clin. Microbiol. 2005a: 43: 906 - 908.Trama J.P., Mordechai E. and Adelson M.E. Detection and identification of Candidaspecies associated with Candida Vaginitis by real-time PCR and pyro sequencing. Mol Cell Probes. 2005b: 19(2): 145-152.Pyrosequencing- Principles And Applications Md. Fakruddin1*, Abhijit Chowdhury1, Md. Nur Hossain1, Khanjada Shahnewaj Bin Mannan2, Reaz Mohammad Mazumdar3 Molecular Biology Vol 2/Issue 2/Apr-Jun 2012Margulies M., Egholm M., Altman W.E., Attiya S. and Bader J.S. et al. Genomesequencing in microfabricated high-density picolitre reactors. Nature. 2005: 437: 376-380.Neve B., Froguel P., Corset L., Vaillant E.,Vatin V. and Boutin P. Rapid SNP allele

frequency determination in genomic DNA pools by pyrosequencing. Biotechniques.2002: 32: 1138-1142.

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