EMULSION PCR,VARIOLA PCR,BRIDGE PCR

EMULSION PCR,VARIOLA PCR,BRIDGE PCR

1. EMULSION PCR  VARIOLA PCR  BRIDGE PCR  M.Vidya kalaivani 12MZBE06 MPhil Biotechnology

2. Principle

3. oil phase composed of 4.5 % Span 80, 0.4% Tween 80, and 0.05% Triton X-100 in mineral oil.

4. The aqueous phase was a PCR mixture . The emulsion PCR mixture was prepared as described below: 0.4 mmol /L of each primer, 3.5 mmol /L MgCl2 , 0.4 mmol /L dATP , 0.4 mmol /L dCTP , 0.4 mmol /L dGTP , 0.4 mmol /L dTTP , 0.125 unit/L Taq DNA polymerase, and 0.01 pmol /ml ssDNA template were added into the Buffer to adjust total volume to 100 ml.

5. Water-in oil emulsions were prepared by adding the ice-cooled PCR reaction mixture (0.1 ml) gradually to the oil phase (0.2ml) in a 2ml round-bottom cryogenic vial whilst the mixture was continuously stirred at 1,500 rpm. For 5 min before PCR cycling.

6. Recovery of the reaction mixture The water-in-oil emulsion from the PCR tube was pooled and spun at 9000 g for 5 min to remove the oil phase, leaving the concentrated emulsion at the bottom of the vial . Two volumes of water-saturated ether were added to one volume of the concentrated emulsion, and the mixture was vortexes and centrifuged briefly to remove the ether phase . The aqueous phase was washed two times with ether and dried at room temperature.

7. DNA ASSAYS Polyacrylamide gel electrophoresis silver staining and Agarose electrophoresis were used for analysis of the PCR products.

8. AM40_261.pdf

9. Why we use this technique????? R ecombination between homologous regions, which results in the formation of chimeric DNA molecules . short fragments tend to be amplified more efficiently in comparison to longer ones Also, highly diverse nucleic acids may not be able to form perfect duplexes following denaturation steps in PCR

10. Conventional PCR Emulsion PCR

11. Applications PCR amplification of random DNA libraries in aptamer selection. Aptamer ????????????? Aptamers are single-stranded DNA or RNA oligonucleotides capable of binding to other target molecules with high specificity, affinity and stability The 454, the Polonator and SOLiD platforms rely on emulsion PCR to amplify clonal sequencing. Next generation sequencing.

12. Bacterial genomes are compacted by Histones Other proteins than histones Supercoiling DNA polymerases DNA topoisomerases CP proteins

13. DNA polymerases join DNA fragments replicate RNA replicate DNA synthesize DNA in 5’->3’ direction synthesize DNA in 3’->5’ direction require a primer to function require nucleotides to function require ATP

14. Bridge pcr Opposite to conventional PCR surface amplification is performed on constant temperature (60°C). Formamide works as a denaturing agent. formamide (at 60°C formamide melts DNA duplexes; equivalent to "denaturation step" in normal PCR); extension buffer (non- denaturating conditions, equivalent to " anealing step" in normal PCR); extension mixture (primer extension step, equivalent to "extension step" in normal PCR);

15. Steps…………. Two PCR primers are attached to the surface of flow cell. One of the primers has a cleavable site (cross on red primer); 

16. Pre amplified library is denaturated in NaOH , then hybridization buffer is added to shift pH to neutral value. Library is loaded into the channel in neutral aquatious solution. DNA molecules can hybridize to the PCR primers . Red primer hybridize with a library molecule on the picture.

17. Elongation reaction E xtension mix (buffer, dNTP's , Taq polymerase) is pumped into a channel. Hybridized primer extended on library molecule . 

18. Formamide Original library molecule is denatured and washed away . 

19. Extension buffer Extended molecule bends and hybridizes to a second PCR primer (forms a bridge). 

20. Extension mixture Extension of hybridized primer. 

21. Formamide washing Two DNA strands are denatured. 

22. B locking of all 3' ends ( ddNTP's and terminal transferase ) to prevent extension of DNA molecules on each other;

23. Applications The focus is on gene discovery or SNP. The sheer volume of data allows greater comparative genomics to be performed e.g. methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus   ( Francois et al , 2007, Future Microbiology) . Re-sequencing allows specific areas to be checked, especially as a cost effective way of discovering SNPs e.g. Bacillus subtilis  re-sequencing identified new mutations and suppressor mutations  ( Srivatsan et al,   2008, PLOS Genetics) .

24.   Surface PCR has lower efficiency if compare with PCR in solution. 35 cycles result in ~1000 copies of the original molecule (in ideal PCR 35 cycles should give ~10 10 x amplification). DNA duplex has high chances to renaturate again instead of hybridising with a new PCR primer . Disadvantage

25. VHF\VARIOLA PCR WHY???????????????????????????????

26. PROBLEM Bioterror attack T he release of number of viruses, such as the variola virus and those causing haemorrhagic fever. The clinical symptoms in the early stages of these viral infections are all very similar and resemble a flu-like illness. Early testing is, therefore, important to establish an accurate diagnosis, but existing diagnostic techniques are either not rapid enough, require high virus concentrations, or lack accuracy .

27. arenavirus  - animal viruses belonging to the family Arenaviridae Junin virus  - the RNA virus that causes Argentine hemorrhagic fever; carried by rats and mice Lassa virus  - the RNA virus that causes Lassa fever Machupo virus  - the RNA virus that causes Bolivian hemorrhagic fever; carried by rats and mice

28. Common methods for laboratory diagnosis of smallpox and VHF are isolation of the virus in cell culture or laboratory animals polymerase chain reaction (PCR ) virus antigen detection electron microscopy as well as detection of specific antibodies in the serum of the patient. Virus detection and isolation in cell culture is still the gold standard for establishing a definitive diagnosis. However, it takes days to weeks to isolate a virus . In contrast to classical diagnostic methods, which are based on the detection and identification of the intact organism, PCR detects the genetic material of a pathogen and thus reduces the contact with infectious material to a minimum. So, even laboratories of lower security level than P4 can perform PCR diagnostics for VHF and smallpox.

29. In order to design reliable primers and hybridisation probes for real-time PCR, additional sequences of the PCR target regions were generated : 30 sequences for Lassa virus GPC gene PCR 22 sequences for Lassa virus L gene PCR 7 sequences for Ebola/Marburg virus L gene PCR 14 sequences for variola virus 14-kDa fusion protein gene PCR.

30. Prototypes of the Orthopox PCR kit Ebola / Marburg virus L gene PCR kit CCHF virus NP gene PCR kit

31. I n order to facilitate wide distribution and safe handling of PCR assays for the most relevant viruses ( variola virus, Ebola and Marburg viruses, Lassa virus, and CCHF virus), prototypes of ready-to-use kits were developed by a small or medium-sized enterprise (SME). They will also be made available to experienced laboratories in Member States (e.g. those of the European Network of Imported Viral Diseases (ENIVD))

32. The sample DNA is placed in a solution containing free nucleotides and the appropriate enzyme The complementary DNA strands are separated New complementary strands are formed using nucleotides from the solution The cycle is repeated(usually 20-30times )

33. At the end of onecycle,two molecules of DNA has been produced from each original molecule. How many DNA molecules will have been produced from one molecule of DNA after 4 complete cycles????? State the enzyme meant by” the appropriate enzyme “in step1.And explain why a specialized form of this enzyme required for PCR?????? The separation of the DNA strands (step2) is normally caused by the enzyme helicase, explain what causes the strand to separation in PCR?????? Suggest one use for PCR technique??????????????

34. a ) 1)24 2)TAQ polymerase 3)Adding formamide 4)DNA amplification b) 1)16 2)DNA polymerase 3)Heat shock/high temperature 4)Forensics increase the amount of DNA present in the sample c) 1)16 2)Topoisomerase 3)high temperature 4)increase the amount of protein present in the sample

35. References Richard Williams1, Sergio G Peisajovich2, Oliver J Miller1,3, Shlomo Magdassi4, Dan S Tawfik2 & Andrew D Griffiths1,3”Amplification of complex gene libraries by emulsion PCR” 2006 Nature Publishing Group http:// www.nature.com/naturemethods. Jay Shendure & Hanlee Ji “Next-generation DNA sequencing” 2008 Nature Publishing Group http:// www.nature.com/naturebiotechnology . Tatjana Schütze a,b , Florian Rubelt”A streamlined protocol for emulsion polymerase chain reaction and subsequent purification” Analytical Biochemistry 410 (2011) 155–157. Srivatsan et al,  2008, PLOS Genetics . http://europa.eu.int/comm/research/fp6/ssp