Pyrosequencing

Hazel Ingram

All Wales Molecular Genetics Laboratory

22/05/2013

• Sequence by synthesis method

• Relies on the release of a pyrophosphate

molecule when 2 nucleotides are joined

together

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.html

Application

• We use pyrosequencing to

determine sequence changes in

tumour cells

• The changes will be present in

only a proportion of the cells,

dependent on the tumour load.

Mutant signal is a % of the

wild-type signal

50% : 50% to ……

Wt

KRAS

Wt

KRAS

Mt

KRAS

Wt

KRAS

Need to detect tumour cells in a background of normal cells

Application

Sample Preparation

1. Amplify region of interest by

PCR, one primer biotinylated

2. Immobilise biotinylated PCR

product onto streptavidin-

coated beads

B S

B

B

B

B

1. Amplify region of interest by

PCR, one primer biotinylated

2. Immobilise biotinylated PCR

product onto streptavidin-

coated beads

Wash Station

http://www.pyrosequencing.com

Sample Preparation

3. Ethanol wash

4. Separate strands by denaturation

5. Wash the immobilised strand

6. Anneal sequencing primer

B S

B S

B S

• 4 enzymes:

– ATP polymerase

– ATP Surfurylase

– Luciferase

– Apyrase

• 2 substrates:

– Adenosine 5’

pyrosulfate (APS)

– Luciferin

Pyro Reaction

http://www.pyrosequencing.com

Pyrogram

Normal

G>T change

Advantages and Limitations

Advantages

• Very sensitive

– Normal PCR -5%

– COLD PCR – 2%

– Sanger Sequencing – 15%

• No fluorescent dyes

therefore no artefacts

• Rapid

Limitations

• Short sequences only

• Homopolymeric regions

difficult to interpret

• Deletions and insertions

difficult to interpret

Thank you for Listening

Thanks to Rachel Butler, tumour team, extraction team and specimen reception staff at All Wales Molecular Genetics laboratory

Contact:

hazel.ingram@wales.nhs.uk