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Mitchell, M. (2006). Pyrimidine-Pyrimidine Transition Polymorphism testing. PHILICA.COM Article number 9.

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Pyrimidine-Pyrimidine Transition Polymorphism testing

Maika Mitchellunconfirmed user (Herbert Irving Comprehensive Cancer Center, Health Sciences, Columbia University)

Published in bio.philica.com

Abstract
Transition is a type of nucleotide-pair mutation involving the replacement of a purine with another purine, or of a pyrimidine with another pyrimidine (e.g. GC with AT). This mutation is much more common than a transversion. The mouth swabs with buccal epithelial cells were air-dried at room temperature, then alkaline extraction was accomplished by soaking with 20 mL 0.2 M NaOH and incubated for 5–6 min at 75°C. The extracts were neutralized with 0.02 M Tris-HCl, pH 7.5,, During PCR, The result of a SNP genotyping assay on the ABI 7900HT will report each sample as homozygous for either allele, or as heterozygous. The raw fluorescent data obtained from each assay, as well as the genotype calls made by the software, are exported to a spreadsheet. This protocol also includes the use of the instrument PyroSequencer (Biotage) and the PSQ 96HS SNP Reagent Kit which contained the enzyme and substrate mixture and nucleotides. Pyrosequencing™ is a gel-free, sequencing-by-synthesis method. In a Pyrosequencing reaction, nucleotide incorporation proceeds sequentially along each DNA template at a given nucleotide dispensation order (NDO) that is programmed into a pyrosequencer. The Pyrosequencer was run in parallel with the ABI 7900HT to show the accuracy of the new method and the efficiency (results for 96 samples in 15-25 minutes).

Article body

 

Reagents/Equipment needed:Reagents: 96 & 384 well clear optical half-skirted plates; TaqManUniversal PCR Master Mix, DNAse-free sterile distilled water, TE Buffer,(10mM Tris-HCl, 1mM EDTA, pH 8.0 made using DNAse-free, sterile filtered water), freshly diluted 10% bleach solution9for decontamination od work areas and swork surfaces inside the hood), VIC(Dye used to label the TaqMan probe that detects allele 1 sequence.), FAM (Dye used to label the TaqMan probe that detects the allele 2 sequence), NFQ (Quencher used at the 3' end of the TaqMan probes), Design Strand (indicates if the probe contains the sequence of the context strand of its reverse complement.), biotinylated labeled primer for the Pyrosequencer PCR reaction, Hair nets, booties( shoe covers), nitrile gloves, disposable 3M sticky mats, disposable lab coats, Pipettement ( P2, P20, P 100, P1000) Pipetteboy and glass tubes up too 15 ml, 8-channel and 12-channel pipettor, conical tubes, ABU 7900HT, Biotage Pyrosequencer, GeneAmp 9700 Thermocycler, centrifuge, mixer/ shaker, heating block(80C), vacuum source:water jet orvacuum pump), 70% ethanol, Streptavdin Sepharose HP( Amersham Biosciences), Vacuum Prep Tool.Controls needed:SNP markers  are publicly available from dbSNP(http://www.ncbi.nlm.nih.gov/SNP/). DNA referenced samples with homozygous, and heterozygous alleles were provided by Dr. Nathan Ellis, Sloan-Kettering Institute/Memorial Hospital (New York CityThe Biotage Pyrosequencer will be used to quantify the complementary DNA strand built up and the nucleotide sequence is determined from the signal peak in the pyrogram(histogram)  and compare the results of the ABI 7900HT cluster plot. These systems will run simultaneously.

Outline of assay:Agarose is used instead of Polyacrylamide gel due to the ease of transfer od DNA. Agarose gel:Pour a gel that is 10cm x 25cm, the quantity of agar needed to pour a 2.0 % agarose gel that is 0.75cm thick.10cm X 25cm X 0.75cm = 187.5 mls.  2.00 X 1.875 = 3.75 grams of agar.

Restriction Enzymes:BbvI(Reagents:1X NE Buffer2: 10mM Tris-HCl,50mM NaCl, 10mM MgCl2, 1.0mM dithiothreitol, pH 7.9 @ 25 C

 

(***Fonts in blue is the area of the polymorphism)

PCR mix to be used on ABI 7900HT: 2.5 µl 10x Buffer (Mg2+ free), 2 µl dNTPs, 1.5 µl MgCl2 (25mM), 16.25 µl ddH2O, 0.25 µl Taq Polymerase (TaKaRa; 5U/µl), 1 µl primer A (10pmol/µl), 1 µl primer B (10pmol/µl), 0.5 µl genomic DNA =25.0 µl.  10x Buffer:, - 100 mM HCl (pH8.3), 500 mM KCl.  dNTPs mixture: 2.5 mM of each Dntp, pH 7-8, solved in water (sodium salts) PCR program ABI 7900HT: 1. 5' 94°C; 2. 30" 94°C; 3. 30" 62°C, 4. 2' 72°C…39 cycles; 6. 5' 72°C; 7. ∞ 4°C. PCR mix to be used on Biotage Pyrosequencer : 1X PCR Buffer, 10 pmoles Forward 5'-GAACAAGCCCAGTAAGCCAAAAA-3' primer, 10 pmoles,5'-TTTTCACGATAGTAACGGTCCTCA-3' reverse primer, 5'-CAGTAAGCCAAAAACCA-3' sequencing primer, 1 U Amplitaq Gold(Perkin Elmer), 0.2 mM of each dNTP, 1.5-3.0 mM MgCl2, 10 ng template. PCR program Biotage Pyrosequencer : 5'95°C; 50 x(95°C 15 sec, ta 30 sec, 72°C 15-30 sec) 72°C  5'…∞ 4°C . Forward primer 5'-biotin-GAACAAGCCCAGTAAGCCAAAAA-3', Reverse primer 5'-biotin-TTTTCACGATAGTAACGGTCCTC-3', Sequemcing primer 5'-biotin-CAGTAAGCCAAAAACCA-3'; ExoSAP-IT* For PCR Product Clean-Up, simple, quick PCR clean-up for Single Nucleotide Polymorphism (SNP) analysis.The method is based on the 5' nuclease activity of Taq DNA polymerase. A standard PCR using primers that will amplify the DNA region containing the SNP of interest is performed on the GeneAmp 9700 thermocycler prior to the SNP genotyping assay, which is prepared via the software package  which comes with the Pyrosequencer and the ABI 7900HT. Then, one set of the amplified DNA product is  put on the ABI 7900 HT and the other set is put on the Pyrosequncer using primers that will amplify the DNA region containing the SNP of interest. Included in the reaction are two allele-specific fluorogenic probes, each consisting of a different fluorescent reporter dye and a fluorescent quencher. In the intact probe, the proximity of the quencher to the fluorphore causes fluorescence resonance energy transfer (FRET), reducing the fluorescence from the reporter dye. During PCR, the 5' nuclease activity of Taq digests the allele-specific probe bound to the region of the SNP, releasing the fluorescent dye from the quencher and allowing generation of a fluorescence signal. Depending on which dye signal is generated, the SNP alleles are determined. If only one dye signal is detected, the SNP is homozygous for the allele corresponding to the allele-specific probe, and if both dyes are detected, then the SNP is heterozygous.Diagram of results: Figure A Sample of the results obtained of a 2.0% Agarose gel using Lambda DNA Marker and BbvI digestion enzyme. Figures B1 & B2 are sample results from SNP Data Analysis Software from the ABI 7900HT; Fig. C Sample results from Pyrosequencer.

Figure A

 

Figure B1.



Figure B2.                                                                                                       




Figure C              

 

Floor Plan Dimensions =(Dimensions are 2.4"  X 5.08" or 24 feet x 50.8 feet)1/2"5ft

                          




 

extracted DNA. (2) Maintain separate, dedicated equipment (e.g., pipettes, microcentrifuges) and supplies (e.g., microcentrifuge tubes, pipette tips) for assay setup and handling of extracted DNA. (3) Wear a clean disposable lab coat and gloves (not previously worn when handling extracted DNA or PCR products) when setting up assays. (4) Change gloves between samples and whenever you suspect they are contaminated. NOTE: PCR technology is extremely sensitive to cross contamination between specimens as well as contamination from outside sources. Gloves should be changed between steps where different specimens are handled or steps involving other potential sources of contamination. (5) Keep reagent tubes and reactions capped as much as possible. (6) Before setting up assays and after handling extracted DNA or PCR products, clean equipment and lab benches with freshly prepared 10% bleach. (7) Do NOT bring extracted DNA or PCR products into the assay setup area. (8) Use aerosol barrier (filter) pipette tips.

Section 1a- Pre-Specimen Processing Area: Samples are brought into the main hallway.  Tech #1 gowns up outside of the lab with: disposable lab coat, booties (shoe covers), hair net and gloves before entering lab space.  These items are also disposed immediately before leaving the lab space in a biohazard receptacle in order to be autoclaved.

 

Section 1b- Clean Room/ Reagent Preparation: Tech #2 gowns up outside of the lab with: disposable lab coat, booties (shoe covers), hair net and gloves before entering lab space.  These items are also disposed immediately before leaving the lab space in a biohazard receptacle in order to be autoclaved.

 

Section 2 - Dark room: Visualization of gels; microscope(s), spectrophotometer; supplies for the instruments in the dark room. Tech #1 or Tech #2 gowns up outside of the lab with: disposable lab coat, booties (shoe covers), hair net and gloves before entering lab space.  These items are also disposed immediately before leaving the lab space in a biohazard receptacle in order to be autoclaved.

 

Section 3 - Specimen Processing/Amplification and storage of processed samples: Tech #1 gowns up outside of the lab with: disposable lab coat, booties (shoe covers), hair net and gloves before entering lab space.  These items are also disposed immediately before leaving the lab space in a biohazard receptacle in order to be autoclaved.

 

Section 4 - Data Analysis: Area to print, view data generated by instruments which are networked to data analysis area

 

Storage Room for reagents / Accessioned Files: Area for paper/CD/floppy disks file(s) of accessioned


Information about this Article
Peer-review ratings (from 1 review, where a score of 100 represents the ‘average’ level):
Originality = 100.00, importance = 136.53, overall quality = 137.00
This Article was published on 11th August, 2006 at 01:18:23 and has been viewed 8715 times.

Creative Commons License
This work is licensed under a Creative Commons Attribution 2.5 License.
The full citation for this Article is:
Mitchell, M. (2006). Pyrimidine-Pyrimidine Transition Polymorphism testing. PHILICA.COM Article number 9.


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1 Peer review [reviewer #2144unconfirmed user] added 17th September, 2006 at 12:45:04

Comprehensive Article.

However, why is Section 4 -Data Analysis only one line? Would be much better if the full data analysis were given.

Another thing is transversion, and transition are American terms, I presume. Some British biologists may be more familiar with the term translation and inversion, and perhaps that could be addressed.

Originality: 4, Importance: 7, Overall quality: 7




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