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SEQUENCING METHODS

SEQUENCING METHODSst
1 generation: 2 methods were invented around 1976,but only one is widely used: the chain-termination method invented by Fred Sanger.
The other method is Maxam-Gilbert chemical degradation method, which is still used for specialized purposes, such as analyzing DNA-protein interactions.st
2 generation: pyrosequencing
Next generation: 454 and Illumina/Solexa sequencing.

A Timeline of The Human Genome YEAR

human genes mappedto a definitechromosome location

years it would taketo sequence the humangenome

1967 none^ sequencing not possibleyet 1977 3 genes mapped^ 4,000,000 years tofinish at 1977 rate 1987 12 genes mapped^ 1000 years to finish at1987 rate 1997 30,000 genes mapped^ 50 years to finish atpresent rate

SANGER^ SEQUENCING
Uses DNA polymerase to synthesize a secondDNA strand that is labeled. DNA polymerasealways adds new bases to the 3’ end of a primerthat is base-paired to the template DNA.
Also uses chain terminator nucleotides:dideoxy nucleotides (ddNTPs), which lack the -OH group on the 3' carbon of thedeoxyribose. When DNA polymerase insertsone of these ddNTPs into the growing DNAchain, the chain terminates, as nothing canbe added to its 3' end.

DNA synthesis requires a 3´-OH to make the nextphosphodiester bond during DNA synthesis normaldNTP

DIDEOXY^ NTPS BLOCK

DNA^ SYNTHESIS^ H

A^ MIXTURE OF DNTP

S AND DDNTPS^ ARE USED IN DNA SEQUENCING

POLYACRYLAMIDE GEL ELECTROPHORESIS IS USED TOVISUALIZE THE RESULTS OF THE SEQUENCING REACTION

AUTOMATED^ DNA

SEQUENCING OUTPUT

4 REACTIONS CARRIED OUT IN ONE TUBE

MAXAM AND^ GILBERT METHOD
This is the best method offered for sequencing of smalloligonucleotides.
Maxam–Gilbert sequencing is a method of DNA sequencingdeveloped by Allan Maxam and Walter Gilbert in 1976.
Maxam–Gilbert sequencing requires radioactive labeling atone 5′ end of the DNA fragment to be sequenced.
The Maxam and Gilbert chemical sequencing method isbased on the ability of different chemical to specificallymodify bases within the DNA molecule.
The chemical specificity of the first reactions are as follows: G: DMS G+A: Formic acid T+C: Hydrazine C: In the presence of NaCl, only C reacts with hydrazine.

PYROSEQUENCING
A luciferase is an enzyme which emits light in thepresence of ATP. Several organisms, such as the American firefly and thepoisonous Jack-o-lantern mushroom, produce luciferases.

DETECTING POLYMERASE ACTIVITY
Pyrophosphate is also known as PPi, also known as“two phosphate groups stuck together”. Duringreplication, each addition of a dNTP releasespyrophosphate
In the reaction mixture, PPi allows adenosinephosphosulfate (APS) to be converted to ATP; this ATPallows luciferase to luciferate (emit light).
Measures strand extension as it happens

PYROSEQUENCING OUTPUT Runs of bases produce higher peaks – for instance, the sequence for (a)is GGCCCTTG. Sample (c) comes from a heterozygous individual (hencethe heights in multiples of ½)

NEXT^ GENERATION

SEQUENCING^ (NGS)

^ Modern high-throughput DNA sequencing technologies
^ parallel, rapid
^ Decreasing price, time, workflow complexity, error rate
^ Increasing data quantity and quality, read lenght (datastorage capacity), repertoire of bioinformatics tools
^ Wide range of applications
^ Starting material:- DNA (DNA-seq)- RNA (RNA-seq)^ Sample^ Library preparation

Bioinformatic Sequencing s