DNA sequencing• Introduction:• Knowledge of DNA sequences has become indispensablefor basic biological research, other research branchesutilizing DNA sequencing, and in numerous applied fieldssuch as diagnostic, biotechnology, forensic biology andbiological systematics. The advent of DNA sequencing hassignificantly accelerated biological research and discovery.The rapid speed of sequencing attained with modern DNAsequencing technology has been instrumental in thesequencing of the human genome, in the human genomeproject. Related projects, often by scientific collaborationacross continents, have generated the complete DNAsequences of many animal, plant, and microbial genomes.
• DNA sequencing includes several methodsand technologies that are used for determiningthe order of the nucleotide bases—adenine,guanine, cytosine, and thymine—in a moleculeof DNA.
Two similar methods used for determining the order of the nucleotidesequences:1. Maxam and Gilbert method2. Sanger method They depend on the production of a mixture of oligonucleotides labeledeither radioactively or fluorescing, with one common end and differing inlength by a single nucleotide at the other end This mixture of oligonucleotides is separated by high resolutionelectrophoresis on polyacrilamide gels and the position of the bandsdetermined
The Maxam-GilbertTechnique• Principle - Chemical Degradationof Purines– Purines (A, G) damaged bydimethylsulfate– Methylation of base– Heat releases base– Alkali cleaves G– Dilute acid cleave A>G
Maxam-GilbertTechnique• PrincipleChemical Degradationof Pyrimidines– Pyrimidines (C, T) aredamaged by hydrazine– Piperidine cleaves thebackbone– 2 M NaCl inhibits thereaction with T
Maxam and Gilbert MethodChemical degradation of purified fragments (chemical degradation)The single stranded DNA fragment to be sequenced is end-labeled bytreatment with alkaline phosphatase to remove the 5’phosphateIt is then followed by reaction with P-labeled ATP in the presence ofpolynucleotide kinase, which attaches P labeled to the 5’terminalThe labeled DNA fragment is then divided into four aliquots, each of which istreated with a reagent which modifies a specific base1. Aliquot A + dimethyl sulphate, which methylates guanine residue2. Aliquot B + formic acid, which modifies adenine and guanine residues3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosineThe four are incubated with piperidine which cleaves the sugar phosphatebackbone of DNA next to the residue that has been modified
Maxam-Gilbertsequencing - modifications
• Chain-termination methods (Sanger method)• The key principle of the Sanger method was the use ofdideoxynucleotide triphosphates (ddNTPs) as DNAchain terminators.• The classical chain-termination method requires1- single-stranded DNA template2- DNA primer3- DNA polymerase4- normal deoxynucleotidephosphates (dNTPs)5-modified nucleotides (dideoxyNTPs) that terminateDNA strand elongation.These ddNTPs will also be radioactively or fluorescentlylabeled for detection in automated sequencingmachines.
In a synthesis reaction, if a dideoxynucleotide isadded instead of the normal deoxynucleotide,the synthesis stops at that point because the3’OH necessary for the addition of the nextnucleotide is absent.
•In the dideoxy method of sequencing, the template DNA that isto be sequenced is mixed with a primer complementary to thetemplate DNA and the four normal dNTPs, one of which isradioactively labeled for subsequent visualization purposes.•This mixture is then splint into four different tubes that arelabeled A, C, G, and T. Each tube is then “spiked” with adifferent ddNTP (ddATP for tube A, ddCTP for tube C, ddGTTfor tube G, or ddTTP for tube T).•DNA polymerase is added and using the DNA template and its’complementary primer, the synthesis of new strands of DNAcomplementary to the template begins.•Occasionally a dideoxynucleotide is added instead of the normaldeoxynucleotide and synthesis of that strand is terminated at thatpoint.
•In the tube containing ddATP, some percentage of newlysynthesized molecules will get a ddATP in each place that there isa T in the template DNA.•The result is a set of new DNA molecules in tube A, each ofwhich ends in an A.•A similar type of reaction occurs in the three other tubes to resultin molecules that end in C, G, and T in tubes C, G, and Trespectively.•After the synthesis reactions are complete, the products of thefour different tubes are loaded onto four adjacent lane of apolyacrylamide gel and the different fragments are separated bysize.•The sequencing gel is able to resolve fragments that differ in sizefrom each other by only one base.
•After electrophoresis to separate the fragments by size,the fragments are visualized to exposing the gel tophotographic film (Remember that one nucleotide wasradioactively labeled).•All fragments in lane A will end in an A, fragments inlane C will all end in a C, fragments in lane G will allend in a G, and fragments in lane T will all end in a T.•The sequence of the DNA is read from the gel bystarting at the bottom and reading upward.
Dideoxy Chain Termination
DNA sequencing: the Sanger methodFour separate polymerizationreactions are performed
DNA sequencing: the Sanger (dideoxy)method
Dye-terminator sequencing–Automated DNA sequencing – in automated DNA sequencing aradioactive deoxynucleotide is not used and all four dideoxyreactions are done in a single tube.– This is possible because each ddNTPs is labeled with a differentflourescent dye.–Therefore the dye present in each synthesized fragmentcorresponds to the dye attached to the dideoxynucleotide thatwas added to terminate the synthesis of that particularfragment.–The contents of the single tube reaction are loaded onto asingle lane of a gel and electrophoresis is done.
– A flourimeter and computer are hooked up to thegel and they detect and record the dye attachedto the fragments as they come off the gel.– The sequence is determined by the order of thedyes coming off the gel.
Semi-Automated Sequencing•thermal cycler•fluorescent ddNTPs•unique spectra•measure intensity ofDNA products on gel
Automated DNA sequencing
DNA SequencingChromatogram file
Automated DNA Sequencing with Fluorescent DyesEach different ddNTP is coupled to a different colored fluorescent dyeddTTP is red; ddGTP is black etc.