Primer designing for pcr and qpcr and their applications
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This document discusses primer design concepts and applications of real-time PCR. It covers guidelines for optimal primer design such as length, GC content, and melting temperature. Longer primers are more specific but risk secondary structure formation. Primer dimers can decrease efficiency. Real-time PCR has applications in biomedical research, genetic disease diagnosis, cancer research, and forensics. It allows monitoring amplification in real-time and precise quantification of DNA or RNA.
Primer designing for pcr and qpcr and their applications
- 1. Primer Designing Concepts & Applications of Real Time PCR Dr Sandeep Agrawal MD Senior Resident & Research Scholar Department of Biochemistry AIIMS, New Delhi
- 2. Primer design guidelines Primer specificity (length 18-24 bases) Primer complexity (avoid repeat regions) Primer GC content (40-60%) Primer melting temperature (55-650C) Amplicon size (100-250 bp) Intra/inter primer complementarity Primer location with respect to template Tm of primer pair should not differ by >50C Length of a primer pair should not differ by >3 bases
- 3. Primer length • There are 4 bases in the DNA molecule A,C,G,T • The probability of encountering any of these bases in the code is 0.25 (1/4) • So let us look at the probability of encountering a particular sequence of bases Event Probability A 0.25 = 0.25 A,T 0.25 x 0.25 = 0.0625 A,T,A 0.25 x0.25 x 0.25 = 0.015625 A,T,A,G,G (0.25)5 = 0.0009765 A,T,A,G,G,T,T,T,A,A,C (0.25)11 = 0.000002384 A,T,A,G,G,T,T,T,A,A,C,C,T,G,G,T (0.25)16 =0.0000000002384 So it is increasingly unlikely that one will get 16 bases in this particular sequence (1 chance in 4.3 billion). In this same way, one can see that as the primer increases in size, the chances of a match other than the one intended for is highly unlikely. Hence, longer the primer, more chance of being unique and highly specific…however…
- 4. What would happen if your primer is too long? If the primer is too long: Annealing temperature will be too high Chances of self-complementarity will be higher Secondary structure formation higher Efficiency might decrease (shorter primers are more efficient) Hence primer length is usually recommended to be 18-24 nt long for human sequences… “Your primer is only as specific as the annealing temperature you select” Even if you design a very specific primer, it would still anneal at non-specific locations if the Tm is too low.
- 5. Primer Dimers • Pair of Primers Primer 1 5’-ACGGATACGTTACGCTGAT-3’ Primer 2 5’-TCCAGATGTACCTTATCAG-3’ • Complementarity of primer 3’ ends 5’-ACGGATACGTTACGCTGAT-3’ 3’GACTATTCCATGTAGACCT-5’ • Results in Primer dimer formation Primer 1 5’-ACGGATACGTTACGCTGATAAGGTACATCTGGA-3’ 3’-TGCCTATGCAATGCGACTATTCCATGTAGACCT-5’ Primer 2
- 6. Primer Dimers • can be due to self complementarity of primers as well.. • usually in the size of 40 -60 bp • decrease the efficiency of amplification of gene of our interest • usually more abundant in NTCs and low template situations •Solutions: • stringent primer design • primer concentration/ template conc. • annealing temperature – gradient PCR • use of HOT-START DNA Polymerase
- 7. Gradient PCR • Select the temperature at which amplification is maximum and primer dimer is not formed/minimum..
- 8. DNA EXON 1 EXON 2INTRON 1 cDNAEXON 1 EXON 2 Genomic DNA vs cDNA
- 9. Primer design Manual design Primer3 Primer BLAST RT-Primer DB PrimerBank Published primers Primer BLAST Primer BLAST
- 17. Applications of PCR & Real Time PCR
- 18. Major areas Biomedical research Diagnosis of genetic diseases Diagnosis of infectious diseases Cancer biology Evolutionary studies Forensic applications
- 19. Biomedical research Amplifying specific DNA segments for cloning Study of gene expression in different paired samples Normal vs tumor Treated vs untreated O hour vs 2 hour Developmental stages Kidney vs liver Study of mitochondrial DNA
- 20. T.A.Brown. Gene cloning and DNAAnalysis: an introduction. Wiley- Blackwell 6th edition.
- 21. T.A.Brown. Gene cloning and DNAAnalysis: an introduction. Wiley- Blackwell 6th edition.
- 23. 1 2 3 4 5 M 6 7 8 9 10 11 12 βActin 172 bp VEGF 226 bp L1 & L6, U87 treated with 1mg/ml BVCZ for 24 hours; L2 & L7, U87 treated with 2mg/ml BVCZ for 24 hours; L3, L4, L5, L8, L9 & L10 cultures of OMEC L11, U87 glioma cell line (positive control) M, 100 bp Ladder RT-PCR analysis to check VEGF mRNA expression in cultivated OMEC
- 24. βActin 172 bp VEGF 226 bp Real Time PCR analysis to check MUC1 mRNA expression in cultivated OMEC
- 28. Diagnosis of genetic diseases
- 31. Diagnosis of HIV infection HIV p24 RNA isolated from blood of 4 suspected HIV patients Reverse Transcription using 500 ng of RNA from all patients PCR for HIV p24 (400 bp amplicon) 25 cycles Copy number of HIV RNA can also be determined by standard curve method
- 33. Evolutionary studies DNA isolated from fossils, PCR amplified, sequenced and analyzed for homology Forensic science Crime scene investigations Paternity testing
- 34. Advantages of Real Time PCR • Amplification can be monitored in real time • Specificity and sensitivity • Detection is capable down to < 2-fold change • Wide dynamic range • No post run processing of products • Confirmation of specific amplification by melt curve analysis
- 35. Disadvantages & limitations of Real Time PCR • Setting up requires high technical skill and support • High equipment cost • Average change in gene expression • Assumptions: housekeeping gene is constantly expressed • Change in mRNA level might not reflect change in protein level