Sanger sequencing Flashcards
DNA sequencing
It is the process of determining the precise order of nucleotides within a DNA molecule
-It determines the sequence of individual genes, full chromosomes or entire genomes of numerous types and species of life, including the human genome.
DNA sequencing history
In 1977, two seperate methods for the large-scale sequencing of DNA were devised:
1. Chemical cleavage method by A.M Maxam and W. Gilbert
2. Enzymatic chain termination method by F. Sanger et, al.
Chemical Cleavage (Maxam and Gilbert)
-Template DNA strand is first separated into single strands
-The single strands are placed in four different test tubes
-The single strands are radio labeled with a 32-phosphate at 5’ end
-Each test tube is treated with a different chemical
-Formic acid only attacks purines and so will be left with C+T strands
-Hydrazine only attacks pyramidines and so will be left with a+g strands
-Dimethyl sulfate will only attack A leaving G
-Alkali and pipe riding will only attack T leaving C
-Placed in agarose gel and read bottom to top
Advantages of chemical cleavage
-No premature termination due to DNA sequencing. So, no problem with polymerase to synthesise DNA
-Long stretches of DNA can be sequenced which can not be done with enzymatic method
Disadvantages of Chemical cleavage
-Not widely used
-Use of radioactivity and toxic chemicals
another name for sanger sequencing
The dideoxy sequencing method
Dideoxy reaction
It is based on DNA polymerisation reqiring a free 3’ OH group to add a 5’ phosphate group from an incoming deoxyribonucleotide triphosphate (dNTP) precursor
ddNTP
Di-deoxyribonucleotide
-It is a nucleotide that lacks a 3’-OH group for a new precursor to be latched on to and so it is used to terminate DNA polymerisation
Sanger sequencing simplifed
A labeled primer is used to iniate DNA synthesis. The addition of four different dideoxy nucleotides randomly arrests synthesis
Sanger sequencing
-ddNTP is in low concentratio to offer a limiting factor to prevent termination from occurring too frequently
-DNA strand is then normally polymerised but randomly terminated at different stages depending on where the ddNTP precursor is incorporated
-ddNTP is radiolbelled and is then identified by polyacrymalide gel electrophoresis and x-ray autoradiography
-However flurosescent ddNTP and laser excitation is more common
-The DNA fragments are then placed in a capillary tube with gel where the fragments are separated and the position of a specific fluorescence marker analysed by a fluorescence detector determines the location and type of base present in a DNA molecule which is translated by a computer
Sanger sequencing requirements
-Fluoresent labelling of ddNTPS enables the sequencing reactions to be conducted in the same container- ‘one-pot’ (tube) reactions
-a thermocylcer- especially to denature Double-stranded DNA
-An RNA primer with a free 3’-OH is required as is a thermostable DNA polymerase and dNTPS
Why is Next-generation sequencing technology useful?
IT saves time because it negates the need for vector recombination, bacterial transformation and cloning as the fragmented DNA is directly sequenced as raw material
Examples of next gen tech
- 454 sequencing
- SOLiD sequencing
3.Illumina (solexa) method
G-banding
-First, cells are taken, usually from blood or tissue samples. These cells are then grown in a lab.
-: The cells are treated with special chemicals that stop them from dividing at specific points in their cell cycle.
- After treatment, the cells are stained with a dye. This dye binds to the chromosomes, but it’s attracted to certain parts more than others. These parts show up as dark and light bands when viewed under a microscope.
Each chromosome has a distinct biding pattern that helps it to be identified as well as if it has abnormalities
