Strand Displacement Amplification

1. Strand Displacement Amplification Presented by Lisa Smith & Apollo Kacsinta

2. Critcal Parameters • Must first denature DNA • Isothermic Temperature • Chlamydia samples must be fresh • 4 – 6 days old max • Takes about 1 hour to process results. • Could be more or less depending on the length of the target sequence

3. Critical Parameters • [dNTP] • Too much dNTPs will lead to easier misincorporation of bases and quench Mg++. • Primer Design • Should have the same annealing temperature. • Should not have complementarities between them. • Should be in excess enough to compete for binding over reanealing of DNA strands.

4. Critical Parameters • [Salt] • Too much will lead to nonspecific binding for probes. • Lowers Stringency • Mg++ is needed for polymerase activity. • Organic Solvents • Ex. Formamide, can lower the stringency of reaction • pH • Optimal pH is dependent on polymerase in use

5. Critical Parameters • [polymerase] • Too much polymerase will allow the polymerase to bind to non specifically bound primers, causing random copied products • Restriction Enzyme • Must be able to cleave DNA at an acceptable rate - concentration • Should have a specific recognition sequence.

6. Controls • Amplification Controls • Monitor Inhibition • Known Positive Control • Known Negative Control • No DNA Control • Much like the controls for PCR

7. SDA • Specifically defined sequence unique to a specific target • SDA is an isothermal process (37°C - 55°C) • Uses series of primers, DNA polymerase and a restriction enzyme to exponentially amplify the unique nucleic acid sequence • SDA can be thought of as occurring in two segments • 1)A target generation phase • 2)Exponential amplification

8. Target Generation • Double stranded DNA is heat denatured creating two single-stranded copies. • Specially manufactured primers combine with DNA polymerase to form altered targets capable of exponential amplification. • Amplification primers; copying the base sequence • Bumper primers; displacing newly created strands

9. The Amplification Phase Step 1 • The exponential amplification process begins with altered targets (single-stranded partial DNA strands with restricted enzyme recognition sites) from the target generation phase.

10. Step 2 • An amplification primer binds to each strand at its complimentary DNA sequence.

11. Step 3 • DNA polymerase uses the primer to identify a location to extend the primer from its 3' end, using the altered target as a template for adding individual nucleotides.

12. Step 4 • The extended primer forms a double-stranded DNA segment containing a complete restriction enzyme recognition site at each end.

13. Step 5 • The restriction enzyme binds to the double stranded DNA segment at its recognition site.

14. Step 6 • The restriction enzyme dissociates from the recognition site after having cleaved only one strand of the double-sided segment, forming a nick.

15. Step 7 • DNA polymerase recognizes the nick and extends the strand from the site, displacing the previously created strand.

16. Step 8 • The recognition site is repeatedly nicked and restored by the restriction enzyme and DNA polymerase with continuous displacement of DNA strands containing the target segment.

17. Step 9 • Each displaced strand is then available to anneal with amplification primers similar to the action in step 2. The process continues with repeated nicking, extension and displacement of new DNA strands, resulting in exponential amplification of the original DNA target.

18. Strand Displacement Amplification

19. Application • Using BD’s DNA amplification technology of Strand Displacement Amplification new and improved test such as the BDProbeTec™ ET System CT and CT/GC Assays have been developed. • This specific test is used for the diagnosis of STIs Chlamydia and Gonorrhea. • The test can by performed on a swab (endocervical/ urethral) from a patient or nonivasively on a urine sample.

20. What is Chlamydia? Chlamydia Trachomatis ~ 1 million bp ; ~895 proteins ; ~939 genes

21. What is Chlamydia? • Gram negative Bacteria • Common STI (Sexually Transmitted Infection) • Curable with Tetracycline type antibiotics • Resistant to Penicillin • Contains RNA and DNA • Obilgate Human Parasite • Can not synthesize it’s own ATP or grow on an artificial medium

22. What is Chlamydia? • Causes infections such as: • Endemic Trachoma • Can lead to blindness • Inclusion Conjunctivitis • Lymphogranuloma Venerem • Swelling of the lymph glands in the groin • Left untreated can lead to infertility

23. 1. Specimen should be processed according to assay specific procedures. 2. Samples are incubated for lysis. 3. Lysed specimen is added to the Priming plate with predispensed dry reagents. 4. Priming plate is then incubated with the Amplification plate. 5. Sample is transferred from the Priming plate to the Amplification plate. 6. Plate sealer is placed onto the Amplification plate. 7. Sealed plate is placed in BDProbeTec ET instrument. Reading takes 60 minutes.

24. Advantages • Offers real-time amplification with simultaneous detection. • The strand displacement amplification products are hybridized with a fluorescent detector probe and are captured by a chemiluminescent assay. • Amplification control is offered with the kit to monitor assay inhibition. • Greater sensitivity than previous techniques (direct probes, enzyme immunoassays (EIA) and culture). • Ready to go predispensed dry reagents.

25. Other Advances • Thermophilic SDA using a higher temp. yields a cleaner, faster product with a higher amplification efficiency. • RT-SDA amplifies RNA targets (HIV gag region) • Size of target sequence has increased can now use a sequence of 2000 bp • BDProbeTec™ ET System for Mycobacterial Assays (i.e. TB)

26. References: • www.bd.com/diagnostics/microservices/innovations/sda/amplification.asp • www.bd.com/clinical/products/bdprobetec/bdprobe_ct2.asp • www.bd.com/clinical/products/bdprobetec/index.asp • www.thedoctorsdoctor.com/diseases/chlamydia.htm#tx