Strand Displacement Amplification

Presented by

Lisa Smith & Apollo Kacsinta

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

Critical Parameters

[dNTP]Too much dNTPs will lead to easier

misincorporation of bases and quench Mg++.

Primer DesignShould 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.

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

Critical Parameters

[polymerase]Too much polymerase will allow the

polymerase to bind to non specifically bound primers, causing random copied products

Restriction EnzymeMust be able to cleave DNA at an

acceptable rate - concentrationShould have a specific recognition

sequence.

Controls

Amplification ControlsMonitor Inhibition

Known Positive Control Known Negative Control No DNA Control Much like the controls for PCR

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

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

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.

Step 2

An amplification primer binds to each strand at its complimentary DNA sequence.

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.

Step 4

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

Step 5

The restriction enzyme binds to the double stranded DNA segment at its recognition site.

Step 6

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

Step 7

DNA polymerase recognizes the nick and extends the strand from the site, displacing the previously created strand.

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.

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.

Strand Displacement Amplification

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.

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

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

What is Chlamydia?

Causes infections such as:Endemic Trachoma

• Can lead to blindness

Inclusion ConjunctivitisLymphogranuloma Venerem

• Swelling of the lymph glands in the groin

Left untreated can lead to infertility

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.

 

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.

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)

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#t

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