3.10 DNA Sequencing

What is DNA Sequencing? Give me some background information.

The process of determining exact order of nitrogenous bases

In July 2000, two major American labs working on Human Genome Project (HGP) announced that 99% of our genes had been sequenced

What method does HGP base itself upon to sequence DNA?

Sanger Dideoxy Method

HGP utilizes what process?

DNA Replication

What 5 components needed for DNA Sequencing?

1. A single stranded DNA template (to be sequenced)

2. Radioactively labeled DNA primer

3. DNA polymerase

4. Nucleoside triphosphates

5. Radioactively labeled dideoxy analogues

What is Step 1 of DNA Sequencing?

DNA to be sequenced is treated so that it becomes single stranded (single stranded DNA template)

What is Step 2 of DNA Sequencing?

Short, single stranded radioactively labeled primer is added to 3’ end of DNA template

What is Step 3 of DNA Sequencing?

• Thousands of identical copies of “primed DNA template” are placed in 4 reaction tubes

• Each contains DNA polymerase I and all four nucleoside triphosphates (dATP, dGTP, dCTP and dTTP)

• Small amount of a different radioactively labeled dideoxy analogue (either ddATP, ddGTP, ddCTP or ddTTP)

What is Step 4 of DNA Sequencing?

Double stranded fragments that form in 4 reaction tubes are separated by length (using gel electrophoresis)

What is Step 5 of DNA Sequencing?

Pattern of bands formed on an autoradiogram can be used to decipher sequence of DNA template

Tell me about Dideoxy Analogues (difference to normal nucleoside+labeled)

• Dideoxy analogues differ from normal nucleoside triphosphates in that they are missing an -OH group on 3’ carbon of their sugar units

• Dideoxy analogues are radioactively labeled

Tell me about Dideoxy Analogues (effect on polymerase+what happens when analogue is added)

• As a result of missing -OH group, DNA polymerase Icannot join next nucleotide to dideoxy analogue

• DNA replication stops when a dideoxy analogue added

Tell me about Dideoxy Analogues (act as a…+why different lengths form)

• Dideoxy analogues act as a chain terminator whenever it joins to DNA template

• Since only a small fraction of available nucleotides in each reaction tube are dideoxy analogues, different lengths of double stranded DNA will form before chain termination occurs

What does using the same gel for all four reaction tubes allow us to do? What can this thing be read from? What does it show?

1. see a pattern that can be used to determine sequence of DNA template

2. Sequence can be read from an autoradiogram

3. Sequence shown on autoradiogram is compliment of

   DNA template