Chain Termination Sequence

Table of Contents
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Definition Uses Facts and Overview of CTS What are Dideoxynucleotides? Manual Sequencing: Step 1 Step 2

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Step 3 Step 4 Step 5 Step 6 Chain Termination Method, and Results Automated Sequencing Relationship of CTS to SBI4U Material Work Cited

Definition
What is Chain Termination Sequencing? The Chain Termination Sequence (CTS) is a DNA polymerase-based technique used in genetics / biochemistry to determine the nucleotide order of a given DNA fragment.

Uses
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To correlate the sequence of a gene with its functions. To re-sequence the same DNA molecule over and over. This is necessary, for example, in the typing of single nucleotide polymorphisms. The CTS method became highly automated due to development during the Human Genome Project, where it was used to sequence fragments of the human genome.

Facts and Overview of CTS
This process is also known as Sanger Sequencing. Developed by Frederick Sanger in 1977 Method:
Electrophoresis can separate DNA molecules Dideoxynucleotide (ddNTP) are used - which stop replication

What are Dideoxynucleotides?

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Dideoxynucleotides are essentially the same as nucleotides except they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH). These modified nucleotides, prevent the addition of further nucleotides. This is because a phosphodiester bond cannot form between the dideoxynucleotide and the next incoming nucleotide, and thus the DNA chain is terminated.

Ma nual Se quencing: St ep 1
 The process is started by synthesizing a chain that is complimentary to the template you want to analyze.

Step 2
 Add a specific primer that you known will

anneal.

Step 3

Divide your sample among four test tubes. One test tube will be used for each specific nucleotide (dGTP, dATP, dCTP, and dTTP). Add DNA polymerase to each tube.

Step 4

Add dideoxunecleotides (ddNTP’s) to all four tubes. Once the ddNTP’s are added, the elongation will stop.

Step 5

Run this on a polyacrylamide gel using one lane per reaction tube (dGTP, dATP, dCTP, and dTTP).

Step 6

To sequence, read the order of bases from the smallest to the largest.

Chain Termination Method, Results

time fragment size

Signal

Automated Sequencing

An automated sequencer is a machine that runs on the same principle as the CTS method. A laser constantly scans the bottom of the gel, detecting bands as they move down the gel. This method is very efficient because it makes it possible for all four reactions to be run in one lane, so you can have huge numbers of reactions on one gel.

Relationship of CTS to SBI4U Concepts
 CTS

relates back to the gel electrophoresis experiment.  In this experiment, we had the same procedure, except the only difference was that we did not use ddNTP’s.  Our experiment, performed in the class, was a basic experiment to prove that DNA sequence can be read using simple methods.

Works Cited

North Dakota State University – Fargo, N.D. (1997). Cloning and Molecular Analysis of Genes. Retrieved December 17, 2005, from http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/cloning/clone6.htm Mama Ji’s Molecular Kitchen. (1999). Sequencing. Retrieved December 16, 2005, from http://lifesciences.asu.edu/resources/mamajis/sequencing/sequencing.html University Of Washington, Pharmaceutics. DNA Sequencing. Retrieved December 16, 2005, from http://depts.washington.edu/pceut/pceut_services/DNASequencing-NCBI.htm

Sarah Obenrader. The Biology Department @ Davidson. (2003). Sanger Method. Retrieved December 19, 2005, from http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Obenrader/s Department of Molecular and Cellular Biology. (2005). Sequencing Principles and Methods. Retrieved December 19, 2005, form http://www.mcb.uct.ac.za/principles%20of%20DNA%20sequencing/sld015.htm Kimball’s Biology Pages. (2003). DNA Sequencing. Retrieved December 19, 2005, from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html