2.  THE DNA MOLECULE.  POLYACRYLAMIDE GEL ELECTROPHORESIS  FLUORESCENCE TECHNOLOGY.  DNA SEQUENCING DEVELOPMENT.  MegaBACE & ABI SYSTEMS.  THE INSTRUMENT COMPARTMENTS.  THE SOFTWARE PROGRAMS.  THE REAGENTS AND KITS.  APPLICATIONS.  SEQUENCING IN LIFE

3. The DNA molecule is made up of two poly nucleotides that form a double helix. Each polynucleotide consists of monomers called nucleotides. A nucleotide consists of a nitrogen base, a pentose and a phosphate group.

4. The portion of this unit with out the phosphate group is called a nucleoside. The bases are pyrmidines and purines, pyrmidines are the cytosine (C) and thyamin (T) purines are adenine (A) and guanine (G). Nucleotides are linked by covalent bonds called phosphodiester linkage between the OH group on the 3′ carbon of one nucleotide and the phosphate on the 5′ carbon of the next. The portion of this unit with out the phosphate group is called a nucleoside. The bases are pyrmidines and purines, pyrmidines are the cytosine (C) and thyamin (T) purines are adenine (A) and guanine (G). Nucleotides are linked by covalent bonds called phosphodiester linkage between the OH group on the 3′ carbon of one nucleotide and the phosphate on the 5′ carbon of the next.

5. POLYACRYLAMIDE GEL ELECTROPHORESIS method for separating proteins and DNA. Solution is applied as bands or spots. Particles migrate in a solvent (buffer). Solvent is supported by an inert polyacrylamide gel When an elecrical field is applied the gel works as molecular seive Molecules are separated by size.

6. Fluorescence is the result of a process that occurs in certain molecules (generally polyaromatic hydrocarbons or heterocycles) called fluorophores or fluorescent dyes. A fluorescent probe is fluorophore designed to localize within a specific region of a biological specimen or to respond to a specific stimulus. Fluorescence is the result of a process that occurs in certain molecules (generally polyaromatic hydrocarbons or heterocycles) called fluorophores or fluorescent dyes. A fluorescent probe is fluorophore designed to localize within a specific region of a biological specimen or to respond to a specific stimulus.

7. The fluorescence labels can be used in direct labeling of nucleic acids by incorporating a nucleotide containing an appropriate fluorophore. A photon of a specific energy is supplied by an external laser source that is absorbed by the fluorophore creating an exited electronic state. when the fluorophore returns to its ground stat, a photon of lower energy and therefore longer wave length than then the excitation energy is emitted. The fluorescence labels can be used in direct labeling of nucleic acids by incorporating a nucleotide containing an appropriate fluorophore. A photon of a specific energy is supplied by an external laser source that is absorbed by the fluorophore creating an exited electronic state. when the fluorophore returns to its ground stat, a photon of lower energy and therefore longer wave length than then the excitation energy is emitted.

8. Manual (Sanger's) method it is determination of nucleotide sequence DNA

9. Automated DNA sequencing

10. Because the slab gel electrophoresis was still manual and needed several steps, modern capillary systems were introduced. Each sample is separated and analyzed in an individual capillary Capillary gel electrophoresis (CGE) can be basically regarded as separation of molecules in a single lane.

11. In CGE fused silica capillaries of 50-100 µm in diameter and 30-80 cm in length are filled with a separation matrix consisting of a gel and electrode buffer. Solution phase DNA molecule are injected into the capillary either by pressure or electrokienetic injection and separated inside the capillary according to their size under very high voltage conditions (5-30 kv). In CGE fused silica capillaries of 50-100 µm in diameter and 30-80 cm in length are filled with a separation matrix consisting of a gel and electrode buffer. Solution phase DNA molecule are injected into the capillary either by pressure or electrokienetic injection and separated inside the capillary according to their size under very high voltage conditions (5-30 kv).

12. The molecules are typically detected using UVlight absorption or laser induced fluorescent detection as they pass a detector at the end of the capillary. The throughput of CE may be enhanced by using multiple capillaries arranged in parallel arrays. The molecules are typically detected using UVlight absorption or laser induced fluorescent detection as they pass a detector at the end of the capillary. The throughput of CE may be enhanced by using multiple capillaries arranged in parallel arrays.

13. ADVANTAGESADVANTAGES Very fast Efficient heat dissipation Use higher run voltages to separate DNA fragments Very short Electrophoresis time

14. Manual low throughput sequencing Labor intensive Relatively expensive Slow-days/months of work Automated high throughput sequencing Machines do the work Relatively Cheap Quick-hours/days of work

15. ABI FROM APPLIED BIOSYSTEMS BIOSYSTEMS Mega BACE 1000 FROM AMERSHAM CAPILLARYELECTROPHORESIS

16. It is a 96 capillary array system based on four color imaging fluorescence detection The MegaBACE 1000 is a high throughput automated gene analysis instrument

17. Light collection system Detection system The electrophoresis compartment http:// www 4.Amersham biosciences.com

18. THE ELECTROPHRESISCOMPATMENT THE ELECTROPHRESIS COMPATMENT The instrument utilizes 6 arrays of 16 capillaries that provide rapid parallel separation of the dye labeled DNA fragments on a total of 96 samples. cathode array stand It is where an array of 16 capillaries assembles into a cathode bar each capillary is then contacted to the surface of the sample plate. capillary detection window each capillary has a clear detection window located at a fixed distance from the sample loading point through which the samples are scanned. anode reservoir holder that contains an anode cover and a plug, and holds six matrix tubes.

19. LIGHT COLLECTION SYSTEM Confocal laser scanning focuses on each sequencing product within the capillary, The excitation light is focused on the sample by an objective lens, and the emitted light is collected by the same lens. Blue laser excites four dyes at 488 nm. Green laser excites four dyes at 532 nm. Green and blue laser mode uses the blue laser to excite two dyes at 488 nm and the green one to excite the other two dyes at 532nm.

20. DETECTION SYSTEM Beamsplitter a beamsplitter separates light by wave length cutoff. Emission filters Two Emission filters separate the overlapping emission from several dyes. Photomultiplier (PMT) collects the filtered light and converts light energy into electrical current.

21. MegaBACE 1000 REAGENTS AND KITS Separation matrix long read matrix with linear Polyacrylamide (LPA) Sequencing reagents DYEnamic ET terminators Thermosequensase DNA polymerase DYEnamic ET primer reagents MegaBACE 1000 REAGENTS AND KITS.

22. To run a sequence with this kit, a reaction premix is combined with a laboratory’s template DNA and primer, the single reaction mixture is thermally cycled. After cycling the reaction products are precipitated with saltAfter cycling the reaction products are precipitated with salt and ethanol or they are passed through a gel-filtration resin and concentrated to remove the unincorporated dye labeled terminators. The reaction products are resuspended in a formamide loadingThe reaction products are resuspended in a formamide loading buffer and then separated and detected in the MegaBACE 1000. buffer and then separated and detected in the MegaBACE 1000.

23. THE SOFTWARE PROGRAMS AND FUNCTION INSTRUMENT CONTROL MANAGER SOFTWARE SEQUENCE ANALYZER SOFTWARE  Provides total control of the system  Controls all the run parameters  Sample injection, electrophoresis,  temperature, gel matrix replacement, and optic configuration.A  gives the power to handle  genomic data  it can analyze data or export data to other files for analysis  retrieve data from as many as 20 runs and analyze up to 1920 samples at a time evaluate the quality of base calling   

24. Up to 6 arrays of 16 capillaries/array for a total of 96 capillaries The capillaries are supplied in pre- assembled arrays of 16 capillaries. 16, 32, 48, 64, 80 or 96 capillaries can be used during a run. Capillary electrophoresis instrument

25. Average read length with dye primer chemistry is 550 bp at 98.5% accuracy With 3-hour run time, sequences of up to 1000 bases is possible. it can produce more than 500 samples per 9-hour day. Ability to sequence 96 samples in just 2 hours and perform up to 9 runs per day

26. Sequencing collection & Analysis software Genotype Fragment Analysis Software

27. The ABI PRISM ® 3100 Genetic Analyzer is an automated capillary electrophoresis system that can separate, detect, and analyze up to 16 capillaries of fluorescently labeled DNA fragments in one run.

29. Complete Automation : The 3100 system offers continuous, unattended operation with automated polymer loading, sample injection, separation and detection, and data analysis. After placing plates on the autosampler and importing sample information, the scientist needs only to select the "Start Run" command. The instrument requires no further attention for up to 24 hours.

30. The capillary array Detection cell Hold the capillaries in place for laser detection Capillary array Enable the separation of the fluorescently labeled DNA fragments by electrophoresis. Filled with polymer. Electrode (anode +) buffer Loding bar (cathode -) Oven Maintains uniform capillaries temperature

31. Capillaries in buffer tank Running and storage position

32. Upper polymer Block Connects the two syringes and the detection end of the capillary array. Array-fill syringe Contains and dispenses the polymer under high pressure to fill the capillaries.(250 µl l) Polymer-reserve syringe Contains and dispenses the polymer that fills the polymer blocks and the array-fill syringe.(5ml)

33. Light collection system As the fragments enter the detection cell, they move through the path of a laser beam. The laser light causes the dye on the fragments to fluoresce. The laser used to excite the dyes is an argon-ion laser. Emission Wavelengths: emission lines are at 488 nm and 514.5 nm ABI 3100

34. DETECTION SYSTEM Laser Excites the attached dye labels as the DNA fragments pass through the detection cell. Transmission Grating Disperses the light by wavelength and asecond set of lenses focuses. CCD camera The CDD camera converts the fluorescence information in to electronic information, which is then transferred to the computer work station for processing by the 3100 data collection software.

35. ABI 3100 REAGENT & KIT REAGENTS & KIT Separation Matrix Polymers Pop-4™ or Pop- 6™ Sequencing Reagents BigDye® Terminator Kit BigDye® primer Kit 5X Buffer

36.  THE SOFTWARE PROGRAMS AND FUNCTION  Data Collection software that controls, monitors, and collects data from the instrument.  Software that automatically extracts and analyzes the data.  A database.  Utilities that enable you to manage the files in the database.  Sample injection, electrophoresis, temperature, gel matrix replacement.  3100 Data Collection Software that enables a user to easily switch between sequencing and genotyping applications without changing polymer type or capillary array Windows NT ® platform, GeneScan ® Analysis Software Macintosh ® platform Software GeneMapper ™ Software v 2.0 Data Collection Software v 1.1

37.  A multi-color fluorescence –based DNA analysis  16 capillaries provides the capability to analyze hundreds of sample per day.  Increases laboratory productivity by process 96- or 384- well plates.  Software suit allows you to generate more meaningful data with less work.  Sample analysis is fully automated  Computer workstation collects and analyzes data from the 3100 Genetic Analyzer.  Hard drive storage 2 drives, Memory 56 MB RAM, monitor  Operating system Microsoft ® Windows ®NT v.4.0, printer, processor. Flexibility and Easy of use of the ABI PRISM® 3100 Genetic Analyzer

38. ABI PRISM ® 3100 Genetic Analyzer Advantages: Capillary Electrophoresis Automation High Throughput Reduced labor costs

39. Higher Sample Throughput A parallel-capillary configuration gives the ABI Prism 3100 Genetic Analyzer the capacity to simultaneously analyze 16 samples with complete automation. Samples are simultaneously injected into the parallel sixteen capillary array in less than 30 seconds The ABI 3100 can process 16 samples every 30 minutes, therefore, we can analyze about 750 samples in a single day.

40. The increased efficiency and sensitivity of a capillary electrophoresis system reduces the amount of DNA required for each sample injection and this, along with labor savings, reduces the overall cost per sample compared to slab gel systems. Reduced Running and Labor Costs The system automatically sequences 16 samples with read lengths greater than 1,000 bp, with 98.5% accuracy in a total run time of 3 hours 40 minutes on the 3100 system.

41. Services & Support Applied Biosystems Web Site To access the Applied Biosystems Web site, go to: http://www.appliedbiosystems.com At the Applied Biosystems Web site, you can:  Search through frequently asked questions (FAQs)  Submit a question directly to Technical Support  Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents  Download PDF documents  Obtain information about customer training  Download software updates and patches In addition, the Applied Biosystems Web site provides a list of telephone and fax numbers that can be used to contact Technical Support.

42. 5789 98OK 984400 ABI3100 GENETIC ANALYZER Flat panel Monitor 3100 v2.0 Data Collection Software Sequence Analysis Software Includes 90 day warranty

43. APPLICATIONS MICROSATELLITEGENOTYPING SNP ANALYSIS The 3100 system can automatically score more than 4,000 genotypes per hour 3100 system can be performed in a 30-minute run time Mapping Set MD10 Kit Used with the 36-cm capillary array, POP-4 polymer, And GeneMapper TMSoftware v 2.0 The ABI SNP genotyping system includes ABI SNaPshot Multiplex Kit and GeneMapper TM Software v 2.0. Used with the 36-cm Capillary array Pop-4 polymer   The MegaBACE SNP genotyping system includes MegaBACE SNP system includes MegaBACE SNP genotyping kit and MegaBACE SNP profiler V 1.0 window based software can deliver >5 760 SNPs per day. The MegaBACE genotyping system genotyping system includes the genotyping test kit includes the genotyping test kit also the MegaBACE genetic profiler score card software automatically processes 2000 microsatellite processes 2000 microsatellite genotypes per 96-well run (14000 genotypes /day)

44. Output The output from the spectral channel is represented by colored traces in graphs called electropherograms. Electropherogram plots relative dye concentration (y-axis) against time (x-axis) for each of the dyes used to label the DNA fragments. The bases correspond to display colors for each type of dye in sequence analyzer. The color of each base is constant. A= GREEN, C= BLUE, T= RED, G= BLACK.

45. Medicine Mutation detection in cancers Pathogen research for treatment of contagious diseases Genetic variations & SNP discovery SNP maps may help scientist to identify the multiple genes associated with cancer, diabetes, vascular and mental diseases. Identify, diagnose and treat genetic diseases

46. Forensic medicine In most cases the purpose of a forensic DNA test is to investigate the possibility that two or more biological samples originated from the same individual Interpretation of a match depends very much on how rare the DNA type is.

47. Forensic medicine Microsatellites are di, tri or tetra nucleotide tandem repeats in DNA sequences. The number of repeats is variable in populations of DNA and within alleles of an individual Microsatellites are used as genetic markers that can be used for genetic diversity, identifying important genetic trait, forensic and paternity studies.

48. Proteomics One of the key practical questions is the role each gene has in synthesizing specific proteins within cells. This field of study, called proteomics, is crucial to applications of DNA sequencing including gene therapy and drug discovery.

49. Species Extinction A team of genome researchers has obtained the first genomic sequences from a woolly mammoth, a mammal that became extinct about 10,000 years ago. The study will clarify the role environmental changes did play in the extinction of an entire species.