Process of determining exact order of nitrogenous bases is called DNA sequencing

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

HGP uses an automated process based on Sanger Dideoxy

Method to sequence DNA

Utilizes process of DNA replication

Five components are needed for this technique:

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

Step 1

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

Step 2

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

Step 3

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

Each contains DNA polymerase 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)

Step 4

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

Step 5

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

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

As a result of missing -OH group, DNA polymerase cannot join next nucleotide to dideoxy analogue

DNA replication stops when a dideoxy analogue added

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

Reaction vessel that contains ddGTP, would produce these fragments of double stranded DNA:

Running results of all four reaction tubes on same gel results in a pattern that can be used to determine sequence of DNA template

Sequence can be read from an autoradiogram

APPLICATION QUESTION FOR TEST

MULTPLE CHOICE WILL AS ABOUT THE PROCESS

what is dd(nucleotide)

what is the cause

what is the point of process

bulk is next part of process

Sequence shown on autoradiogram is compliment of DNA template

The data shown is the complimentary strand

1. name the complimentary strand

2. name the template strand

for the test

only questions 1-19

cloning is NOT on the test

process where you have tot do something (gel electrophoresis,, pcr, transcription and translation)

only ask more in multiple choice

draw meselson-stahl

copare pcr and dna replication

WILL BE ON TEST: write out dna replication

what type of mutation