DNA Sequencing Scramble

Class instructions

In this lesson • DNA sequencing is a powerful method for analyzing DNA

• Ratio of chain terminators (ddNTPs) is important• Importance of complementary base pairing• DNA polymerase only works in the 5’- 3’ direction• Electrophoresis separates DNA fragments by size• Chain terminators stop the reaction & visualize the result

• Why fluorescent labels are used more than radioisotopes

• Software (bioinformatics) is used to line up sequences

The devil is in the detail!

• The 5’ prime and 3’ prime ends of the bases must be round the right way!

IMPORTANT: Do not take

bases apart!!!

Correct base pairing is critical!

• Green (Guanine) pairs with yellow (Cytosine)

• Blue (Adenine) pairs with orange (Thymine)

Bases with white sugars are CHAIN TERMINATORS

or dideoxynucleotides (ddNTP)

This is the DNA we are going to sequence

This is the primer that will begin the reaction

Annealed primer

Automated sequencing

• A whole class activity• There are four pots, one for each nucleotide, each with 15 normal and 5 ddNTPs

• Produce a copy of our template using the labeled nucleotides

• Align resulting products in one column to read sequence

The activity• Each student takes it in turns to synthesise a new strand

• Randomly select a nucleotide from each pot• Add them in the 5’ to 3’ direction• If you pull out a ddNTP, your chain is finished, it is the next student’s turn

The result - electrophoresis

• Line up all the fragments from longest to smallest in one column

• Read the sequence from the smallest fragment upwards

Automated Sequencing Results

G

C

A

C

T Rea

d s

equ

ence

in t

his

dir

ecti

on

Electrophoresis gel is read by a laser - printout produced

Read sequence in this direction

Sanger Sequencing

• Original method of sequencing• Chain terminating nucleotides are radioactively labeled

• Sequencing takes place in four separate tubes, each with one ddNTP

• All four results are then run in four separate lanes on a gel, separated by size

The activity• As before put the template at front of the class

• Denature the two strands and anneal the primer:

• Then line up the automated sequencing products in size order using one lane per base.

Sanger Sequencing Result -

ElectrophoresisRead sequence in

this direction

• Line up products in 4 columns – one for each base:

How an electrophoresis gel would look

G A T C

Read sequence in this

direction

The difference between two methods

• Original Sanger method requires four lanes because all fragments are the same colour.– Radioactive labels = hazardous

• Automated sequencing can be run in one lane because each fragment is coloured differently.– Coloured labels = safe