DNA sequencing and PCR

Question 1

What kind of nucleoside is used for sanger sequencing and why?

Answer 1

2’, 3’-Dideoxyribonucleotide triphosphate, NTP - RADIOACTIVELY LABELLED

• they are chain terminators of DNA replication
• create fragments of differing lengths
• have no 3’ OH group, so when added to DNA no more nucleotides can be added

Question 2

What is the difference between a nucleotide and a nucleoside?

Answer 2

nucleoside: base and pentose sugar

nucleotide: base, pentose sugar and phosphate groups

Question 3

What is the process of Sanger Sequencing?

Answer 3

Add:
- a single stranded template of DNA
- a primer complementary to part of the template
- DNA polymerase
- a pool of normal deoxynucleotides (dATP, dTTP, dGTP, dCTP)
- a small proportion of radioactively labelled ddATP

1) template + primer + DNA pol + dNTPs
2) split solution into 4 for each base
3) add appropriate ddNTP to each one
4) separate by electrophoresis by size
5) autoradiograph (like x-ray + photographic film) and read sequence from the bottom up

Question 4

What is the process of automated dideoxy sequencing?

Answer 4

1) template + primer + DNA pol + ALL dNTPs + ALL FLUORESCENT ddNTPs IN ONE TUBE
2) dye present in each fragment corresponds to the ddNTP
3) electrophoresis + read with lasers