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Sequencing of DNAKey to Genomics

R. SaicharanDept. of Biotechnology,SNIST, Hyderabad Reasons for DNA Sequencing

(practical day-to-day)checking mutationschecking constructs in cloningconstructing phylogeniesfinding genes

DNA SequencingReasons for DNA Sequencing

(philosophical, pioneering)Deciphering “code of life”Understanding organisms, physiology, evolution, disease, cellular behaviour, etc.

Why and WhatSequencing” means finding the order of nucleotides on a piece of DNA .Nucleotide order determines Amino acid order, and by extension, protein structure and function (proteomics)An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect in a plant or animal.

Why and What?Understanding a particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatmentDNA technology is also extended to environmental, agricultural and forensic applications.

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Methods of sequencing

Historically there are two main methods of DNA sequencing:Maxam & Gilbert, using chemical sequencingSanger, using dideoxynucleotides.Modern sequencing equipment uses the principles of the Sanger technique

Founders of Sequence Tech

Fred SangerMRC-Cambridge

Wally GilbertHarvard

How it Works

The fundamental idea behind both methods is the same. One needs a known starting point on the DNA and then a method to detect where each base is positioned on the DNA strand. Both methods terminate a DNA strand at a given nucleotide by either synthesizing to the base or by chemically breaking the DNA.

Chemical or Maxam-Gilbert Sequencing Methods

Around 1977, Wally Gilbert (Harvard) and his technician Allan Maxam developed chemical sequencing. In the late 70s, it was the method of choice.Uses a chemical reaction specific for each nucleotide.

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Maxam and Gilbert Sequencing- One st r a nd o f D N A i s l abe l ed on t he end w i th r ad i oac ti v it y- Ca r ry ou t 5 s e par a t e ch e m ic a l r eac ti on s wh i ch a tt ack spec ifi c group s on

ba s esG , G+ A , C+ T , C, and A>C .

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Reading the Sequence

In the 70's the chemical method of sequencing was widely used because it was more reproducible and available than the enzymatic method.

The enzymatic method required single stranded DNA-templates, oligonucleotide primers and pure enzymes which were not readily available at the time.

However once M13 and phagemid vectors were developed to produce single stranded DNA templates, oligonucleotidesynthesis procedures were worked out to make inexpensive DNA primers.

Also, the price of enzymes dropped and became more available, making the enzymatic method of sequencing easier and more reliable.

The chemical method is now mostly used to sequence regions of DNA that are troublesome by the other method, or to assay for proteins that bind to specific sites on the DNA.

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The Sanger Technique

Uses dideoxynucleotides (dideoxyadenine, dideoxyguanine, etc)These are molecules that resemble normal nucleotides but lack the normal -OH group.

Because they lack the -OH (which allows nucleotides to join a growing DNA strand), replication stops.

Normally, this wouldbe where another phosphateIs attached, but with no -OHgroup, a bond can not form and replication stops

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The Sanger method requires

Multiple copies of single stranded template DNAA suitable primer (a small piece of DNA that can pair with the template DNA to act as a starting point for replication)DNA polymerase (an enzyme that copies DNA, adding new nucleotides to the 3’ end of the templateA ‘pool’ of normal nucleotidesA small proportion of dideoxynucleotides labeled in some way ( radioactively or with fluorescent dyes)

The template DNA pieces are replicated, incorporating normal nucleotides, but occasionally and at random dideoxy (DD) nucleotides are taken up.This stops replication on that piece of DNAThe result is a mix of DNA lengths, each ending with a particular labeled DDnucleotide.Because the different lengths ‘travel’ at different rates during electrophoresis, their order can be determined.

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Originally four separate sets of DNA, primer and a single different DD nucleotide were produced and run on a gel.Modern technology allows all the DNA, primers, etc to be mixed and the fluorescent labeled DDnucleotide ‘ends’ of different lengths can be ‘read’ by a laser.Additionally, the gel slab has been replaced by polymer filled capillary tubes in modern equipment.

Sequencing Hardware

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Step 1- Before submission for sequencing DNA purity & concentration is checked with the ‘Nanodrop’

A Nanodrop readout of known concentration to be run as a control

Step 2 -Samples are received and stored in the refrigerator and a request filed

Cost?

Cost is dependant on a number of factors but typically in 2003:Each tube of sample DNA costs $27 to run.An entire set of 96 tubes from one source (the capacity of the present equipment) costs $960.The methods used will readily analyze DNA fragments of 500-1000 bases in length, depending on the quality of DNA usedNote – the dye alone to run 5000 reactions costs $61,000

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Samples arrive in Eppendorf tubes

Step 3 - paperwork. Each request is assigned a ‘well’ in the sample tray and volumes of primers, water, dye, etc are calculated. A typical ‘run’ has samples from a number of researchers

Step 4- Samples are agitated then centrifuged in an Ultracentrifuge to be sure they are in the bottom of their Eppendorf tubes.

Step 5 - Reagents, etc

Each reaction requires several reagents:Specific primers for the DNA in questionFluorescent Dye attached to DD nucleotides (Big Dye)Deionised waterDNA polymeraseAdditionally, a ‘control’ sample of a known DNA is prepared so it can run at the same time as the experimental DNA.

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Micropipettors come in a range of sizes. They have disposable tips that hold tiny amounts of required reagents.

Step 6 - Preparing the wells

The Sample wells are loaded with DNA to be sequenced. Great care needs to be taken to ensure that each sample goes into its assigned well.Reagents are added (water, dye, primers) in required amountsThe sample wells are ‘spun’ to ensure that the DNA and reagents are mixed and at the bottom of the sample wells.

Sample tray and micropipettor. Each tray holds 96 samples

Step 7 - The samples are run through a cycle sequencing process to get the fluorescent dyes incorporated by the DNA.The DNA and reagents are alternately heated and cooled over a2 1/2 hour period.

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Step 8 - Sample purification to get rid of extra dye and salts

Unincorporated dye and salts can interfere with DNA analysis and need to be removedSamples are centrifuged, precipitated with 95% ethanol, centrifuged again, and drainedThe process is repeated with 70% ethanolDry samples are either analyzed immediately or stored in the dark (light degrades the fluorescent dyes used)Just before sequencing formamide is added to ensure that the DNA remains linear

Entering data from the record sheet into the Sequencer software programme

Step 10- The sequencer is warmed up, reagents are refreshed and the sample tray is inserted Inside the sequencer

Capillary tubes

Sample tray goes here

Reagents

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The Sequencer Apparatus

Each sample tray has 96 wells (1 per sample), and the analyzer (3100 model) has the capacity to analyze 16 wells at a timeRobotic apparatus moves the sample tray so each of the 16 wells is in contact with a separate capillary tube filled with a polymer - this replaces a lane on an electrophoresis gelLabeled DNA from that well moves up the capillary tube, with smaller labeled fragments moving more quickly than longer ones