Different platforms of Sequencing...Part I

Now a days commercially there are 4 types of Platform used for sequencing purpose.

• Sanger Sequencing
• 454 Pyrosequencing
• Solexa/illumina Sequencing
• SOLiD Sequencing

Among them the last 3 are used for high-throughput sequencing , so are the candidates for Genome Sequencing Projects.

Let's see first how the Sanger Sequencing works!!

Sanger Sequencing:

During 1974 two independent methods for Sequencing was invented initiating a new era of Molecular Biology named as Sanger Method and Maxam-Gilbert Method . Sanger Sequencing is also called dideoxy sequencing as here dideoxynucleotides are used along with normal nucleotides. This method is popular for it's simplicity ... the dideoxynucleotides acts as chain terminator as these bases don't have 3 ' OH group thus can't form the phosphodiester bond with upcoming nucleotide.

So here four types of dideoxynucleotides are used in 4 different tubes along with 4 types of normal nucleotide. Say in the G tube only dideoxyGuanine is added to normal A , T , C and G .

In Sequencing reaction the DNA sample should first be denatured by heat to get single strand. The primer is annealed at the 5 ' end and then the termination reaction starts in each tube. This amplified products are subjected for electrophoresis and are separated depending on fragment gradient. After electrophoresis the gel looks like something the picture bellow.

Now a day the 4 type of dideoxynucleotides are labelled with different dyes and can be differentiated by different emission spectra. So all the reactions can go on in a single tube with proper reaction condition in thermal cycle and thus can be subjected for electrophoresis in single lane instead of four!!

The image bellow shows a standard sequence chromatogram using Sequence Scanner by Applied Biosystem.

For the termination reaction Big dye reagent of Applied Biosystem is the most popular one with their many Capillary Sequencer. I have a plan to write about Capillary Electrophoresis Sequencing in details afterwards.

Many commercial institute provide the facility of Sanger sequencing. The output format of Sanger Sequence file is generally in Standard Chromatogram Format (.SCF) or ABI sequencer data files (.ABI and .AB1) or PHRED output files (.PHD). They can be assembled by various assembly software like DNA Dragon , Phrap , CLC Bio , etc. DNA Dragon is free for all and Phrap is only for academic purpose.