DNA Sequencing

What is DNA sequencing?

Determines the nucleotide bases in a DNA sample. Uses special nucleotides called dideoxynucelotides (ddNTP)

How do dideoxynucleotides from normal deoxynucleotide triphosphate?

• dideoxynucleotide differs from normal deoxynucleotide triphosphate in that they lack a hydroxyl group (OH) needed for further attachments

What happens when a dideoxynucleotide is added to a forming strand of DNA?

• Elongation of that strand is ended

• The next deoxynucleotide is not able to bond to that strand

Requirements for DNA sequencing

• Single stranded DNA

• Free nucleotides (A,C,T,G)

• Dideoxynucleotides labelled with different coloured fluorescent dye for easy detection

• Primers

• TAQ DNA polymerase

• DNA Sequencing Machine

DNA Sequencing Steps

1. DNA extracted from cell nucleus and broken up into multiple pieces using enzymes

2. Multiple copies of this DNA are produced

3. Double stranded DNA seperated into single strands

4. Primers added which bind to one strand and initiate the replication process

5. The second strand of DNA is recreated by adding complementary nucleotides (deoxynucleotide triphosphate) in a correct sequence using TAQ polymerase

6. A terminator nucleotide (dideoxynucleotide) stops the nucleotide sequence

7. The strands are compared to determine the nucleotide sequence