Primer Designing Concepts& Applications of Real Time PCRDr Sandeep Agrawal MDSenior Resident & Research ScholarDepartment of BiochemistryAIIMS, New Delhi

Primer design guidelinesPrimer 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 complementarityPrimer location with respect to templateTm of primer pair should not differ by >50CLength of a primer pair should not differ by >3 bases

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 ProbabilityA 0.25 = 0.25A,T 0.25 x 0.25 = 0.0625A,T,A 0.25 x0.25 x 0.25 = 0.015625A,T,A,G,G (0.25)5 = 0.0009765A,T,A,G,G,T,T,T,A,A,C (0.25)11 = 0.000002384A,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…

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.

Primer Dimers

• Pair of PrimersPrimer 1 5’-ACGGATACGTTACGCTGAT-3’Primer 2 5’-TCCAGATGTACCTTATCAG-3’

• Complementarity of primer 3’ ends 5’-ACGGATACGTTACGCTGAT-3’ 3’GACTATTCCATGTAGACCT-5’

• Results in Primer dimer formationPrimer 1 5’-ACGGATACGTTACGCTGATAAGGTACATCTGGA-3’ 3’-TGCCTATGCAATGCGACTATTCCATGTAGACCT-5’

Primer 2

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

Gradient PCR

• Select the temperature at which amplification is maximum and primer dimer is not formed/minimum..

DNAEXON 1 EXON 2INTRON 1

cDNAEXON 1 EXON 2

Genomic DNA vs cDNA

Primer design

Manual design

Primer3

Primer BLAST RT-Primer DBPrimerBankPublished primers

Primer BLASTPrimer BLAST

Applications of PCR & Real Time PCR

Major areas

Biomedical researchDiagnosis of genetic diseasesDiagnosis of infectious diseasesCancer biologyEvolutionary studiesForensic applications

Biomedical research

Amplifying specific DNA segments for cloningStudy 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

T.A.Brown. Gene cloning and DNA Analysis: an introduction. Wiley- Blackwell 6th edition.

T.A.Brown. Gene cloning and DNA Analysis: an introduction. Wiley- Blackwell 6th edition.

1 2 3 4 5 M 6 7 8 9 10 11 12

βActin172 bp

VEGF226 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

βActin172 bp

VEGF226 bp

Real Time PCR analysis to check MUC1 mRNA expression in cultivated OMEC

Diagnosis of genetic diseases

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

Evolutionary studies

DNA isolated from fossils, PCR amplified, sequenced and analyzed for homology

Forensic science

Crime scene investigationsPaternity testing

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

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