Real Time PCR & its applications <ul><li>  </li></ul><ul><li>Dinesh Kumar </li></ul><ul><li>M.Sc. Biotech(BHU), Ph. D.  Bi...
Why to measure gene expression? <ul><li>Chargoff's law two laws </li></ul><ul><li>Traits vary </li></ul><ul><li>Structural...
Why to quantify mRNA?  Gene expression studies
Measurement of mRNA <ul><li>Cell- 100 mRNA of gene A </li></ul><ul><li>Extract- Northern blotting-densiometry </li></ul><u...
How to maximize the accuracy? <ul><li>Use of PCR </li></ul><ul><li>Measure the threshold (Ct) </li></ul><ul><li>Compare wi...
How to quantify mRNA? <ul><li>Northern blotting:   </li></ul><ul><li>In situ  hybridization:  </li></ul><ul><li>RNAse prot...
How real time PCR machine works?   01/06/10 Dinesh-NBAGR-Real time  Time PCR Applications-Summer Training-01-June-2010
How the fluorescence monitoring is done in real time PCR machine? 01/06/10 Dinesh-NBAGR-Real time  Time PCR Applications-S...
Advantages of qPCR   <ul><li>Wide dynamic range of quantification (7–8 log decades). </li></ul><ul><li>High technical sens...
Limitations of qPCR <ul><li>PCR product increases exponentially. </li></ul><ul><li>Variation increases with cycle number. ...
Primers for RT <ul><li>Specific primers: decreases background priming: </li></ul><ul><li>Random hexamers: maximizes the nu...
Quantification of mRNA-  in vitro 01/06/10 Dinesh-NBAGR-Real time  Time PCR Applications-Summer Training-01-June-2010
RNA-poor quality 01/06/10 Dinesh-NBAGR-Real time  Time PCR Applications-Summer Training-01-June-2010
Primer/Probe designing for qPCR <ul><li>Primer Express </li></ul><ul><li>Molecular Beacon-3 </li></ul><ul><li>Primer 3.0 <...
Common qPCR Instruments <ul><li>ABI Prism 7700, 7000 (Perkin-Elmer–Applied Biosystems, Foster City, CA, USA) </li></ul><ul...
Basic chemistries used in qPCR <ul><li>1. DNA-binding dyes  </li></ul>SG (S) will not bind to ssDNA and the intensity of f...
Advantages of DNA-binding dyes  <ul><li>This method obviates the need for target-specific fluorescent probes, but its spec...
Melt curve analysis 01/06/10 Dinesh-NBAGR-Real time  Time PCR Applications-Summer Training-01-June-2010
Limitations of DNA-binding dyes  <ul><li>any double-stranded DNA generates fluorescence   </li></ul><ul><li>amplification ...
2. Molecular beacons  <ul><li>  </li></ul><ul><li>The hairpin structure causes the MB to fold when not hybridized, bringin...
Limitations of molecular beacons  <ul><li>design of the hybridization probe. </li></ul><ul><li>interfere with the hybridiz...
Hybridisation probes  <ul><li>fluorescence resonance energy transfer ( FRET )  </li></ul>Resonance energy (E) transfer is ...
Advantages of Hybridization probes: <ul><li>increases specificity  </li></ul><ul><li>probes are not hydrolyzed, fluorescen...
Hydrolysis probes   Energy (E) emitted by the donor (D) fluorophore is absorbed/quenched by the acceptor (A) fluorophore. ...
Limitations of Hydrolysis probes <ul><li>reduces the processivity of the polymerases </li></ul><ul><li>less efficient   wh...
Amplicon design  <ul><li>< 100 bp,  no longer than 80 bp  are found to be ideal </li></ul><ul><li>as long as 400 bp   </li...
Primer & probe designing <ul><ul><li>Primers </li></ul></ul><ul><ul><ul><li>15-30 bp in length  </li></ul></ul></ul><ul><u...
Data analysis in qPCR <ul><li>  What is the Ct-value?   </li></ul>01/06/10 Dinesh-NBAGR-Real time  Time PCR Applications-S...
Amplification Plots Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time  Time PCR Application...
Dissociation Curve Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time  Time PCR Applications...
Standard Curve Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time  Time PCR Applications-Sum...
What are the reaction efficiency, amplification and slope and how is it calculated? smaller the Ct value ( y  intercept) i...
Amplification Plots Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time  Time PCR Application...
Factors affecting reproducibility of Ct <ul><li>When work with low copy number. </li></ul><ul><li>Short amplicons have gre...
Why and how to Normalize expression data?   <ul><li>any variation in the amount of starting material  </li></ul><ul><li>a ...
Absolute Quantification   C (cRNA} µg/l N (molecules perµl)  =  __________________  X 182·5×10 13 K (fragment size /bp)  W...
Relative and Comparative Quantification <ul><li>relative to an active reference control (normalizer or house keeping genes...
Relative and Comparative Quantification <ul><li>Advantages  </li></ul><ul><li>No standard curve is required. </li></ul><ul...
Mutation/allele detection   <ul><li>A single base mismatch under the probe  </li></ul><ul><li>Decreases the Tm by as littl...
Applications <ul><li>Quantification </li></ul><ul><ul><li>Absolute </li></ul></ul><ul><ul><ul><li>Gene Expression </li></u...
Multiplexing <ul><li>4 channel machine </li></ul><ul><li>Single PCR tube-data of 4 different genes </li></ul><ul><li>Probl...
Ten most common Real-Time qPCR Pitfalls <ul><li>Poor primer and probe design </li></ul><ul><li>Poor quality RNA </li></ul>...
Reaction set up 01/06/10 Dinesh-NBAGR-Real time  Time PCR Applications-Summer Training-01-June-2010
01/06/10
Real time monitoring 01/06/10 Dinesh-NBAGR-Real time  Time PCR Applications-Summer Training-01-June-2010
<ul><li>  </li></ul><ul><li>Dinesh Kumar </li></ul><ul><li>Scientist (Animal Biotechnology) </li></ul><ul><li>Small Rumina...
Upcoming SlideShare
Loading in …5
×

Real time pcr applications-training-june 2010

7,790 views

Published on

For training of NBAGR-Summer training

Published in: Technology, Business
1 Comment
4 Likes
Statistics
Notes
No Downloads
Views
Total views
7,790
On SlideShare
0
From Embeds
0
Number of Embeds
13
Actions
Shares
0
Downloads
599
Comments
1
Likes
4
Embeds 0
No embeds

No notes for slide
  • From Stratagene Site
  • Real time pcr applications-training-june 2010

    1. 1. Real Time PCR & its applications <ul><li>  </li></ul><ul><li>Dinesh Kumar </li></ul><ul><li>M.Sc. Biotech(BHU), Ph. D. Biotech(BHU), PDF(USA) </li></ul><ul><li>Senior Scientist (Animal Biotechnology) </li></ul><ul><li>Genes & Genetic Resources Molecular Analysis Lab </li></ul><ul><li>National Bureau of Animal Genetic Resources </li></ul><ul><li>Karnal-132001 </li></ul><ul><li>E-mail: dineshkumarbhu@gmail.com, +91-94161-11753(SMS only) </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    2. 2. Why to measure gene expression? <ul><li>Chargoff's law two laws </li></ul><ul><li>Traits vary </li></ul><ul><li>Structural variants of gene </li></ul><ul><li>Functional variants of gene </li></ul><ul><li>Structural genomics to functional genomics </li></ul>
    3. 3. Why to quantify mRNA? Gene expression studies
    4. 4. Measurement of mRNA <ul><li>Cell- 100 mRNA of gene A </li></ul><ul><li>Extract- Northern blotting-densiometry </li></ul><ul><li>Extract-cDNA-template-PCR-threshold </li></ul><ul><li>Accuracy in number? 40, 50, 60 molecules </li></ul><ul><li>Accuracy loss </li></ul><ul><ul><li>Extraction </li></ul></ul><ul><ul><li>Transfer/blotting </li></ul></ul><ul><ul><li>Hubridisation/washing </li></ul></ul><ul><ul><li>Signal/noise </li></ul></ul><ul><ul><li>Reading/manual error </li></ul></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    5. 5. How to maximize the accuracy? <ul><li>Use of PCR </li></ul><ul><li>Measure the threshold (Ct) </li></ul><ul><li>Compare with house keeping genes. </li></ul><ul><li>Relative gene expression. </li></ul><ul><li>Straight line equation(range) </li></ul><ul><li>PCR efficiency 100% </li></ul><ul><li>How to do all these? </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    6. 6. How to quantify mRNA? <ul><li>Northern blotting: </li></ul><ul><li>In situ hybridization: </li></ul><ul><li>RNAse protection assays: </li></ul><ul><li>RT-PCR </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010 Gene XXXXx XXXX X GAPDH +/+ +/+ 1 2 3 kb 0.0 0.5 1.0 1.5 2.0 2.5 Thymus Spleen Lymph Kidney
    7. 7. How real time PCR machine works? 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    8. 8. How the fluorescence monitoring is done in real time PCR machine? 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    9. 9. Advantages of qPCR <ul><li>Wide dynamic range of quantification (7–8 log decades). </li></ul><ul><li>High technical sensitivity (< 5 copies). </li></ul><ul><li>High precision (< 2% CV of CT values). </li></ul><ul><li>No post-PCR steps like running of gel or sequencing etc. </li></ul><ul><li>Non-specific amplification can be detected by melt curve analysis of PCR products. </li></ul><ul><li>Minimized risk of cross contamination. </li></ul><ul><li>High throughput. </li></ul><ul><li>Multiplex approach possible. </li></ul><ul><li>Saves lot of time. </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    10. 10. Limitations of qPCR <ul><li>PCR product increases exponentially. </li></ul><ul><li>Variation increases with cycle number. </li></ul><ul><li>Increased variation after transformation to linear values. </li></ul><ul><li>Overlap of emission spectra. </li></ul><ul><li>Maximal four simultaneous reactions limit the bacterial diagnostics. </li></ul><ul><li>Increased risk of false negative results where parasite genome changes very fast. </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    11. 11. Primers for RT <ul><li>Specific primers: decreases background priming: </li></ul><ul><li>Random hexamers: maximizes the number of mRNA molecules thus often overestimate mRNA copy numbers. </li></ul><ul><li>Oligo-dT primers: maximizes the number of mRNA molecules </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    12. 12. Quantification of mRNA- in vitro 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    13. 13. RNA-poor quality 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    14. 14. Primer/Probe designing for qPCR <ul><li>Primer Express </li></ul><ul><li>Molecular Beacon-3 </li></ul><ul><li>Primer 3.0 </li></ul><ul><li>OLIGO </li></ul><ul><li>Tm calculator </li></ul><ul><li>m-fold </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    15. 15. Common qPCR Instruments <ul><li>ABI Prism 7700, 7000 (Perkin-Elmer–Applied Biosystems, Foster City, CA, USA) </li></ul><ul><li>Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany) </li></ul><ul><li>Biorad Instruments- i cycler </li></ul><ul><li>Corbett Research-Rotor Gene </li></ul><ul><li>Opticon-II, Chromo 4 (MJ Research) </li></ul><ul><li>Stratagene (Mx4000) </li></ul><ul><li>Techne-Cephid-Smartcycler </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    16. 16. Basic chemistries used in qPCR <ul><li>1. DNA-binding dyes </li></ul>SG (S) will not bind to ssDNA and the intensity of fluorescent signal is low b) SG (S) binds to dsDNA the fluorescent signal intensity (E) increases 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    17. 17. Advantages of DNA-binding dyes <ul><li>This method obviates the need for target-specific fluorescent probes, but its specificity is determined entirely by its primers. </li></ul><ul><li>Simple and fast. </li></ul><ul><li>Melt curve analysis ensures specificity of amplified PCR products. </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    18. 18. Melt curve analysis 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    19. 19. Limitations of DNA-binding dyes <ul><li>any double-stranded DNA generates fluorescence </li></ul><ul><li>amplification of a longer product will generate more signal than a shorter one. </li></ul><ul><li>amplification efficiencies different- inaccurate result. </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    20. 20. 2. Molecular beacons <ul><li>  </li></ul><ul><li>The hairpin structure causes the MB to fold when not hybridized, bringing quencher and fluorophore dyes in close proximity causing quenching of fluorescence. When hybridized, the fluorophore and quencher are separated resulting in increased fluorescence. </li></ul><ul><li>Advantages of Molecular Beacons: </li></ul><ul><li>specificity </li></ul><ul><li>non-PCR amplification assays </li></ul><ul><li>Limitations </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    21. 21. Limitations of molecular beacons <ul><li>design of the hybridization probe. </li></ul><ul><li>interfere with the hybridization </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    22. 22. Hybridisation probes <ul><li>fluorescence resonance energy transfer ( FRET ) </li></ul>Resonance energy (E) transfer is low when the probes are not hybridised. Hybridisation of the probes brings the donor (D) and acceptor (A) fluorophores into close proximity resulting in increased resonance energy transfer. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    23. 23. Advantages of Hybridization probes: <ul><li>increases specificity </li></ul><ul><li>probes are not hydrolyzed, fluorescence is reversible :allows the generation of melting curves. </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    24. 24. Hydrolysis probes Energy (E) emitted by the donor (D) fluorophore is absorbed/quenched by the acceptor (A) fluorophore. The polymerase exonuclease activity separates the D fluorophore from the A fluorophore by hydrolysis resulting in an increase in fluorescent signal. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    25. 25. Limitations of Hydrolysis probes <ul><li>reduces the processivity of the polymerases </li></ul><ul><li>less efficient when mutation-specific RT-PCR </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    26. 26. Amplicon design <ul><li>< 100 bp, no longer than 80 bp are found to be ideal </li></ul><ul><li>as long as 400 bp </li></ul><ul><li>minimum 63 bp . </li></ul><ul><li>Primer and probe design </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    27. 27. Primer & probe designing <ul><ul><li>Primers </li></ul></ul><ul><ul><ul><li>15-30 bp in length </li></ul></ul></ul><ul><ul><ul><li>G/C content of 20-80% </li></ul></ul></ul><ul><ul><ul><li>Avoid primer dimers. </li></ul></ul></ul><ul><ul><ul><li>The Tm should be within 2°C </li></ul></ul></ul><ul><ul><ul><li>Purify by gel electrophoresis or HPLC </li></ul></ul></ul><ul><ul><ul><li>Optimize concentrations by performing matrix of 50nM, 300nM and 900nM fo the forward and reverse primers. This range allows the matching of the experimental Tm of the primers by +/- 2 ° </li></ul></ul></ul><ul><ul><li>Probes </li></ul></ul><ul><ul><ul><li>20-30 bp in length </li></ul></ul></ul><ul><ul><ul><li>G/C content of 20-80% </li></ul></ul></ul><ul><ul><ul><li>Tm 7-10 ° higher than primers </li></ul></ul></ul><ul><ul><ul><li>To maximize signal or reporter vary the probe concentration between 5-400nM </li></ul></ul></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    28. 28. Data analysis in qPCR <ul><li>  What is the Ct-value? </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    29. 29. Amplification Plots Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    30. 30. Dissociation Curve Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    31. 31. Standard Curve Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    32. 32. What are the reaction efficiency, amplification and slope and how is it calculated? smaller the Ct value ( y intercept) in the regression equation, the greater the sensitivity of the RT-PCR reaction. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    33. 33. Amplification Plots Kumar SYBR Green, 12-18-2003, 10Hr 00Min.mxp.txt 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    34. 34. Factors affecting reproducibility of Ct <ul><li>When work with low copy number. </li></ul><ul><li>Short amplicons have greater reproducibility. </li></ul><ul><li>It is always better to use replicates for greater accuracy. </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    35. 35. Why and how to Normalize expression data? <ul><li>any variation in the amount of starting material </li></ul><ul><li>a cellular RNA that serves as an internal reference against which other RNA values can be normalized. </li></ul><ul><li>housekeeping genes (HKGs) </li></ul><ul><li>GAPDH ,  -actin , rRNA </li></ul><ul><li>H3 and cyclophilin, tubulin, micro-globulin etc </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    36. 36. Absolute Quantification   C (cRNA} µg/l N (molecules perµl) = __________________ X 182·5×10 13 K (fragment size /bp) Where, N= gene molecules per µl, C = concentration of the cRNA inµg/l, K= fragment size or bp, 182·5×10 13 Avagadro constant. 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    37. 37. Relative and Comparative Quantification <ul><li>relative to an active reference control (normalizer or house keeping genes) </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    38. 38. Relative and Comparative Quantification <ul><li>Advantages </li></ul><ul><li>No standard curve is required. </li></ul><ul><li>More wells available for samples. </li></ul><ul><li>Disadvantages </li></ul><ul><li>More optimization and validation required </li></ul><ul><li>If dynamic range of HKG and target genes are not of similar magnitude it needs correction factors. </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    39. 39. Mutation/allele detection <ul><li>A single base mismatch under the probe </li></ul><ul><li>Decreases the Tm by as little as 3 0 C for G–T </li></ul><ul><li>By 10 0 C for A–C mismatches. </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    40. 40. Applications <ul><li>Quantification </li></ul><ul><ul><li>Absolute </li></ul></ul><ul><ul><ul><li>Gene Expression </li></ul></ul></ul><ul><ul><ul><li>Viral, Fungal, Bacterial Load </li></ul></ul></ul><ul><ul><ul><li>Genomic </li></ul></ul></ul><ul><ul><ul><li>Mitochondrial DNAs </li></ul></ul></ul><ul><ul><ul><li>Quality Control </li></ul></ul></ul><ul><ul><li>Relative and Comparative </li></ul></ul><ul><ul><ul><li>Gene expression </li></ul></ul></ul><ul><ul><ul><li>Microarray validation </li></ul></ul></ul><ul><li>Detection </li></ul><ul><ul><li>Sequence Detection </li></ul></ul><ul><ul><ul><li>Allele discrimination </li></ul></ul></ul><ul><ul><ul><li>SNP analysis </li></ul></ul></ul><ul><ul><ul><li>Zygosity testing </li></ul></ul></ul><ul><ul><ul><li>Zoonotic agents </li></ul></ul></ul><ul><ul><ul><li>GMO screening </li></ul></ul></ul><ul><ul><ul><li>Residual disease </li></ul></ul></ul><ul><ul><ul><li>Splice variants </li></ul></ul></ul><ul><ul><ul><li>Chromosomal translocations </li></ul></ul></ul><ul><ul><ul><li>Methylation </li></ul></ul></ul><ul><ul><li>Fluorescence Detection </li></ul></ul><ul><ul><ul><li>Isothermal signal amplification </li></ul></ul></ul><ul><ul><ul><li>Plate reader functionality </li></ul></ul></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    41. 41. Multiplexing <ul><li>4 channel machine </li></ul><ul><li>Single PCR tube-data of 4 different genes </li></ul><ul><li>Problems </li></ul><ul><li>Advantages </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    42. 42. Ten most common Real-Time qPCR Pitfalls <ul><li>Poor primer and probe design </li></ul><ul><li>Poor quality RNA </li></ul><ul><li>Not using “master mixes” </li></ul><ul><li>Introducing cross contamination </li></ul><ul><li>Not using a “-RT”control </li></ul><ul><li>Using and inappropriate normalization control </li></ul><ul><li>Melt curve analysis not done when using SYBR Green </li></ul><ul><li>Baseline and threshold not set correctly </li></ul><ul><li>Efficiency of the reaction is poor </li></ul><ul><li>Using an inappropriate range for standard curve </li></ul>01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    43. 43. Reaction set up 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    44. 44. 01/06/10
    45. 45. Real time monitoring 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010
    46. 46. <ul><li>  </li></ul><ul><li>Dinesh Kumar </li></ul><ul><li>Scientist (Animal Biotechnology) </li></ul><ul><li>Small Ruminant Molecular Genetics Lab </li></ul><ul><li>DNA Fingerprinting Unit </li></ul><ul><li>National Bureau of Animal Genetic Resources </li></ul><ul><li>Karnal-132001 </li></ul><ul><li>E-mail:dineshbhu@rediffmail.com, 094161-11753 </li></ul>Thanks ! I am thankful to Dr J.M.Reecy and his group for Making me happen to learn a b c of real time in real spirit! Thank you to every one here ! 01/06/10 Dinesh-NBAGR-Real time Time PCR Applications-Summer Training-01-June-2010

    ×