Automated DNA Sequencing

LECTURE 7:

Biotechnology; 3 Credit hours

Atta-ur-Rahman School of Applied Biosciences (ASAB)National University of Sciences and Technology (NUST)

Automated DNA Sequencing

Introduction

• Fluorescence based cycle sequencing system is an extension and refinement of Sanger dideoxy sequencing.

It consist of following steps

1. Template preparation 2. Cycle sequencing 3. Purification after cycle sequencing4. Capillary electrophoresis5. Data analysis

Introduction

• Like Sanger sequencing, fluorescence based cycle sequencing requires

• a DNA template, • a sequencing primer, • a thermal stable DNA polymerase, • nucleotides (dNTPs), • dideoxynucleotides (ddNTPs), • and buffer.

• It uses fluorescent dyes to label the extension products and the components are combined in a reaction

Labeling of DNA

• Automated cycle sequencing procedures incorporate fluorescent dye labels using

1. Dye labeled dideoxynucleotide triphosphates (dye terminators)

2. Dye labeled primers (dye primers)

• Both chemistries use four different dyes

Dye Terminator Chemistry

• With dye terminator chemistry, each of the four dideoxynucleotide terminators is tagged with a different fluorescent dye

• One reaction is performed, containing the enzyme, nucleotides, and all dye-labeled dideoxynucleotides

• The products from this reaction are injected into one capillary

Dye Terminator Chemistry

Dye Primer Chemistry

• With dye primer chemistry, four separate tubes of sequencing primer are each tagged with a different fluorescent dye.

• Four separate reactions are performed, each containing the enzyme, nucleotides

• Specific dye labeled sequencing primer, and either A, C, G, or T dideoxynucleotides

• The products from these four reactions are then combined and injected into one capillary

Dye Primer Chemistry

Detection of Resolved Fragments

Detection of Resolved Fragments

Detection of Resolved Fragments

Detection of Resolved Fragments

Fluorescent sequencing compared with radioactive sequencing

Short Gun Sequencing