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of the Experiment: 04

Date: 31.07.2011

Name of the Experiment: DNA Sequencing by ABI 3130. Introduction: In 1977, dideoxy chain-termination method of DNA sequencing was devised by Frederick Sanger. This method uses ddNTPs, which causes termination of reaction during DNA strand elongation producing fragments of varying lengths. Next electrophoresis approach is used to arrange these fragments according to their size, which reveals the nucleotide composition of the sample. After Frederick Sanger scientists had developed several other processes of DNA sequencing that vary from each other on the aspect of time, complexity, hazard, accuracy, efficiency and cost. On continuation of this development, in 21st century various biotech companies had developed automated sequencing machines that are highly efficient, less time consuming, more accurate and cost effective. ABI 3130 genetic analyzer is such kind of machine for DNA sequencing. Surprisingly, its working method is based on the dideoxy chain-termination method, which was developed about three decades ago. It can work in an automated fashion without any kind of human effort. It can perform PCR, capillary electrophoresis, fluorescent ddNTP detection, data arrangement and storage. The person just needs to load the samples and collect the results. The 3100 Genetic Analyzer, a multi-color fluorescence-based DNA analysis system with 16 capillaries operating in parallel, offers high-quality data and efficient sample processing for the busy research lab. One can run a wide variety of sequencing and fragment analysis applications including microsatellite analysis, AFLP, LOH, SNP validation, and SNP screening -- as well as de novo sequencing and resequencing (mutational profiling). The full range of applications can be run on a single polymer and capillary array meaning one can run mixed applications on one plate. The software even includes tools to assist with regulatory and compliance requirements. Machine parts: Figure: ABI 3130 genetic analyzer

Machine Part

Function

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No. of the Experiment: 04

Date: 31.07.2011

Anode reservoir.

buffer Contains 16 mL of 1X running buffer.

Buffer and water Each contains 16 mL of 1X running buffer or water. reservoirs (four) Autosampler Holds the sample plates and reservoirs and moves to align the samples, water, or buffer with the capillaries. Enables the separation of the fluorescent-labeled DNA fragments by electrophoresis. It is a replaceable unit composed of 4 capillaries.

Capillary array

Detection cell block Holds the capillaries in place for laser (argon) detection. andheater Lower polymer block Oven Contains the buffer valve, anode electrode, and anode buffer reservoir. Maintains uniform capillary array temperature.

Polymer pump (PDP) Pump block

delivery Pumps polymer into the array and performs maintenance procedures

Includes the displacement pump chamber, piston waterseal, array attachment point (array port), and connection to the lower polymer block through the interconnect tube

Used reagents: Separations Matrix The 3130 POP-7, POP-6, and POP-4 polymers can be used on the 3130xl System as the separation matrix. Within it, the DNA runs through arrays. Before each run, the capillaries are automatically cleaned. Big dye: It contains:

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Date: 31.07.2011

DNA polymerase dNTPs Fluorescent labledddNTPs. 100% ethanol 3M Na-acetate Hi-Di formamide.

Procedure: 1. 2. 3. 4. The DNA sample to be sequenced was taken in a PCR tube. Big Dye was added in the sample. PCR was performed using only the forward primer. 24 l 100% ethanol,1 l 3M Na-acetate(pH 4.9-5.2) were mixed with each sequencing PCR product (5l) into fresh eppendorf tube and waited for 5 min after 10 sec short-spin, and then centrifuged at 4000rpm for 15 min. Supernatant was removed by using micro-pipette without touching the inner wall of eppendorf. 100 l of 80% ethanol was added and centrifuged at 4000 rpm for 13 min. Ethanol was removed by using micro-pipette without touching the inner wall of eppendorf. The opened eppendorf was air-dried in dark place for 15-20min. The air-dried eppendorf was placed at 40 C. 10 l Hi-Di formamide was added into dried, purified sequencing PCR product before loading into the sequencing analyzer. The purified products were placed on the tray in the sequencer machine. The machine ran for 1 hour and simultaneously sequence four samples.

5. 6. 7. 8. 9. 10. 11. 12.

Result: The result was shown with a graph. Along the peaks of the graph, the sequence was also written. Part of the graph is shown below:

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No. of the Experiment: 04

Date: 31.07.2011

Discussion: Knowledge of DNA sequence has become indispensable for biological research, other research branches utilizing DNA sequencing and in numerous such as diagnostic, biotechnology, forensic biology. Advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of human genome, in the HGP (Human Genome Project). The high demand of low cost sequencing has driven the development of high throughtput sequencing technologies that parallel the sequencing process producing thousands or millions of sequences at once. High-throughtput technologies are intended to lower the cost of DNA sequencing beyond what is possible with dye-terminator methods. Few bases in the first and last part of the sequence cannot be sequenced efficiently by the machine. This is because too much small sequences at start cause problem during analysis and at the later portion, many of the reagents are used up and not available for reactions. In out experiment, big dye was used, which provide the require reagent components for the sequencing reaction in ready reaction, pre-mixed format. This dye is suitable for performing fluorescence based cycle sequencing reaction and single strand or double strand DNA template, on PCR fragments and on large template (for example, BAC clones).

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