Pyromania USAFSAM “EPI Lab” MSgt Stephen Christian, USAF, MSgt, MT (ASCP) Lucinda Sinclair, GS-11 1 Distribution Statement A: Approved for Public release;

Pyromania

USAFSAM “EPI Lab”

MSgt Stephen Christian, USAF, MSgt, MT (ASCP)Lucinda Sinclair, GS-11

1

Distribution Statement A: Approved for Public release; distribution is unlimited. 311 ABG/PA No. 10-080, 12 Mar 2010

Team Aerospace Begins Here!

Overview

Introduction to Influenza

Why\What to Sequence

Sequencing (Pyro versus Sanger)

Pyrosequencing for influenza antiviral resistance markers (Tamiflu resistance in 2009 A (H1/N1)

2


Influenza

http://www.ncbi.nlm.nih.gov/genomes/FLU/

3


Flu Mortality

1918

1957

1968

annual

4

1918 = H1N1 50,000,000 deaths

1957 = H2N2 1,000,000 deaths

1968 = H3N2 500,000 deaths

Annual flu 250,000 deaths

H5N1 annual 25 - 75

Types of influenza

2009 A/H1N1

A,B, and C (Epidemics = A &B)

Pandemics = A only


Influenza Genome

PB2

PB1

PA

HA

NP

NA

M

NS

5


HA1 region of HA gives the most bang for the buck.

Why/What to Sequence

PB2

PB1

PA

HA

NP

NA

M

NS

13,588 bp’s

6

AmantadinePresence/Absence of Antiviral resistance markers

Tamiflu


Nucleic Acid Extraction

Traditional RT PCR

Sequencing

7

Sequencing (Pyro versus Sequencing (Pyro versus Sanger) Sanger)


Sanger Sequencing

Sequencing by Chain Termination

Extract Nucleic Acid

Produce amplicon

Clean Amplicon

Big Dye Rxn (Chain Termination)

Clean Big Dye Rxn

Electrophoresis of Big Dye (Sequencer)

Analyze Sequence Data

8


Produce Amplicon (HA1)

Template (Amplicon)

9


Clean Amplicon (HA1)

10

Template (Amplicon)

Template (Amplicon)

Clean PCR Product

Amplicon

(dATP’s)

(dGTP’s)

(dCTP’s)

(dTTP’s)

Polymerase Salts

Buffers


Template (Amplicon)

Primer Primer

Primer

Primer

Primer

Primer

Primer

dNTP’s

A (dATP’s)

G(dGTP’s)

C (dCTP’s)

T (dTTP’s)

ddNTP’s

A

G

C

T

Big Dye Reaction


Big Dye Reaction

12

Template (Amplicon)


Clean Big Dye Reaction

13


Electrophoresis

14


Results from Electrophoresis

15


Sequence Analysis

Build Single Contig

Analyze constructed nucleotide sequence (Protein)

Relate to current vaccine strain on phylogenetic tree

16


PyrosequencingPyrosequencing

17

Sequencing by Addition

Extract Nucleic Acid

Produce amplicon

Pyrosequencer




2. Immobilize biotinylated PCR products onto streptavidin-coated beads

3. Separate strands by denaturation in NaOH

5. Anneal sequencing primer

96 samples are processed in parallel

4. Wash /neutralize the immobilized strand

1. Amplify relevant region by PCR (100 - 300 bp), using 1 biotinylated primer

Simple Sample Preparation


1.) Hybridize sequence primer along with DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5’ phosphosuflate (APS) and luciferin.

Polymerase

Apyrase

sulfurylase

luciferaseAPS

luciferin

Polymerase



dNTP’s added one at atime

Apyrase

sulfurylase

luciferaseAPS

luciferin

Polymerase

2.) Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi.



Apyrase

sulfurylase

luciferaseAPS

luciferin

Polymerase




Apyrase

sulfurylase

luciferaseAPS

luciferin

Polymerase


PPi



Apyrase

sulfurylase

luciferaseAPS

luciferin

Polymerase

ATP sulfurylase converts PPi and APS to ATP. Luciferase converts luciferin and ATP to oxyluciferin which generates light read by a camera.

+ PPi = ATP



Apyrase

sulfurylaseluciferase

APS

luciferin

Polymerase


+ PPi = ATP

oxyluciferin



Hybridize sequence primer, DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5’ phosphosuflate (APS) and luciferin.

Add one of four dNTP’s, DNA polymerase incorporates dNTP’s (if it is complementary) releasing PPi.


Apyrase degrades dNTP’s and ATP, when complete next dNTP is added.

As process continues, the correct DNA sequence is built up and displayed.


Team Aerospace Begins Here! 27


5 Sanger sequencing steps

VS.

2 Pyrosequencing steps


Neuraminidase Pyrosequencing

29

Team Aerospace Begins Here! 30

Questions

Documents

Pyromania USAFSAM “EPI Lab” MSgt Stephen Christian, USAF, MSgt, MT (ASCP) Lucinda Sinclair, GS-11 1 Distribution Statement A: Approved for Public release;