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Fundamentals of Bioinformatics Different methods of sequencing

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Tiêu đề Fundamentals of Bioinformatics Different Methods of Sequencing
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Bài giảng chủ đề Tin Sinh Học Những điều cơ bản về Tin Sinh Học bằng tiếng Anh. Trong bài giảng này, chúng ta tìm hiểu về những khái niệm cơ bản trong tin sinh học và một số phương pháp giải trình tự (Sequencing methods) cơ bản

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 Bioinformatics is the application of

information technology

◦ to the management of biological data

◦ to the study of biological problems

◦ to store, retrieve and assist in understanding

biological information

Bioinformatics Biology Medicine

Computer science Maths/physics

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 Maxam/Gilbert chemical sequencing

 Automated sequencing

 Nanospore sequencing

 Cheap sequencing

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Maxam-Gilbert sequencing is performed by

chain breakage at specific nucleotides

DMS

G

G G G

FA

G A

G G A G A

A

H

C

T T C T C

C T

H+S

C C C

C

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Sequencing gels are read from bottom to top (5′ to 3′).

G G+A T+C C

3′

A A G C A A C G T G C A G

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Template area to be sequenced

-3′ OH TCGACGGGC…

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 With addition of enzyme (DNA polymerase), the primer is extended until a ddNTP is

encountered

 The chain will end with the incorporation of the ddNTP

 With the proper dNTP:ddNTP ratio, the

chain will terminate throughout the length

of the template

 All terminated chains will end in the ddNTP added to that reaction

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 The collection of fragments is a sequencing ladder.

 The resulting terminated chains are resolved

by electrophoresis

 Fragments from each of the four tubes are

placed in four separate gel lanes

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Sequencing gels are read from bottom to top (5′ to 3′).

G A T C

3′

G G T A A A T C A T G

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 Cycle sequencing is chain termination

sequencing performed in a thermal cycler

 Cycle sequencing requires a heat-stable DNA polymerase

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 Fluorescent dyes are multicyclic molecules

that absorb and emit fluorescent light at

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 In dye primer sequencing, the primer

contains fluorescent dye–conjugated

nucleotides, labeling the sequencing ladder

at the 5′ ends of the chains

 In dye terminator sequencing, the

fluorescent dye molecules are covalently

attached to the dideoxynucleotides,

labeling the sequencing ladder at the 3′

ends of the chains

ddA

ddA

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T

G

T

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G A T C

The DNA ladder is resolved in one gel lane or

in a capillary

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 The DNA ladder is read on an

electropherogram

Capillary Slab gel

5′ A G T C T G

Electropherogram

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5′ A G T C T G 5′ A G( T/ A ) C T G 5′ A G A C T G

T/T T/A A/A

capillary instruments.

sequence in text and electropherogram form.

 Peak patterns reflect mutations or sequence changes.

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 Scientists needed $3 billion and 13 years to sequence the three billion base pairs encoded in

 By 2011, eight years after that first project was completed, the cost of sequencing a human genome had fallen to $5,000, in a process that took just a few weeks.

engineer and the founder of the biotech company Ion Torrent, unveiled an approach that is faster and cheaper still He says his machine will be able to sequence a human genome, some 3.2

$1,000

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 Genetic information is stored in the order or sequence of nucleotides in DNA.

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 SeqVerter

 DNAclub

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