1. Design and Applications in Molecular Biology Research: Primer Design
Module 11

2. Acknowledgment Jennifer M. Anderson, Ph.D.
Li Liu, M.D. Interdisciplinary Center for Biotechnology Research, University of Florida, June 10, 2003
Worachart Sirawaraporn, Dept of Biochemistry, Mahidol University, January 28, 2005
Zhang Univ of Western Ontario, CS 461b/661b: Bioinformatics Tools and Applications on Jan 20, 2005

3. General knowledge of DNA replication

4. PCR

5. Considerations About Primers
Characteristics of primers:
Thoughts on primer design:
Uniqueness
Specificity
Specific for the intended target sequence (avoid nonspecific hybridization)
Length
Base Composition
Internal Stability
Stability
Form stable duplex with template under PCR conditions
Melting Temperature
Annealing Temperature
Internal Structure
Compatibility
Primers used as a pair shall work under the same PCR condition
Primer Pair Matching

6. Where do primers come from ?
Chemically synthesized as ssDNA
Typically nucleotides in length
The nucleotide sequence determined by
- prior knowledge of target DNA sequence
- reverse translation of protein sequence

7. Primer design elements
Primer length
GC%
Annealing
3’ complementary between primers
G&C runs at the 3’ end
Palindrome sequences

8. Good Primer’s Characteristic
A melting temperature (Tm) in the range of 52°C to 65°C
Absence of primer dimerization capability
Absence of significant hairpin formation (>3 bp)
Lack of secondary priming sites
Low specific binding at the 3' end (ie. lower GC content to avoid mispriming)

9. Uniqueness NOT UNIQUE! UNIQUE!
There shall be one and only one target site in the template DNA where the primer binds, which means the primer sequence shall be unique in the template DNA.
There shall be no annealing site in possible contaminant sources, such as human, rat, mouse, etc. (BLAST search against corresponding genome)
Template DNA 5’...TCAACTTAGCATGATCGGGTA...GTAGCAGTTGACTGTACAACTCAGCAA...3’
TGCTAAGTTG
CAGTCAACTGCTAC
TGCT AGTTG A
Primer candidate 1 5’-TGCTAAGTTG-3’
NOT UNIQUE!
Primer candidate 2 5’-CAGTCAACTGCTAC-3’
UNIQUE!

10. Length
Primer length has effects on uniqueness and melting/annealing temperature.
Longer the primer = more unique
Longer the primer = higher melting/annealing temperature.
Generally speaking, the length of primer has to be at least 15 bases to ensure uniqueness. Usually, we pick primers of bases long. This range varies based on if you can find unique primers with appropriate annealing temperature within this range.

11. Base Composition
Base composition affects hybridization specificity, melting/annealing temperature and internal stability.
Random base composition is preferred. We shall avoid long (A+T) and (G+C) rich region if possible.
Template DNA 5’...TCAACTTAGCATGATCGGGCA...AAGATGCACGGGCCTGTACACAA...3’
TGCCCGATCATGCT
GCCCG CAT T
T AT GC
Usually, average (G+C) content around 50-60% will give us the right melting/annealing temperature for ordinary PCR reactions, and will give appropriate hybridization stability. However, melting/annealing temperature and hybridization stability are affected by other factors, which we’ll discuss later. Therefore, (G+C) content is allowed to change.

12. Melting Temperature
Melting Temperature, Tm – the temperature at which half the DNA strands are single stranded and half are double-stranded.. Tm is characteristics of the DNA composition; Higher G+C content DNA has a higher Tm due to more H bonds.
Calculation
(Base Composition): Tm = *(%GC) - 600/length
Other more accurate methods are available.

13. Primer Annealing Temperature
Simple Method (Thein and Wallace 1986):
A and T = 2o C
G and C = 4o C
So, add up the number of A and T in the primer and multiply by 2 and then add up the number of G and C and multiply by 4. Add these two number together to get the approximate annealing temperature.
Exercise:
If the following was your primer set (below) using the method above, what would be the annealing temperature you would use for the reaction:
Forward ATG TGT GAC GAT GAG GTT GC
Reverse TGG CTG GGG CAT TGA AGG TC

14. Stringency in Primer Annealing
Stringency determines the specificity of the amplified DNA product. Tanneal is the most significant factor affecting the stringency in primer annealing.
Tanneal : too low  less stringent  primer matches elsewhere
too high  more stringent  primer may fail to match
Other factors:
GC%: GC pairs are more stringent than AT paris
Salt & Buffer

15. Internal Structure
If primers can anneal to themselves, or anneal to each other rather than anneal to the template, the PCR efficiency will be decreased dramatically. They shall be avoided.
However, sometimes these 2 structures are harmless when the annealing temperature does not allow them to take form. For example, some dimers or hairpins form at 30 C while during PCR cycle, the lowest temperature only drops to 60 C.

16. Primer Pair Matching
Primers work in pairs – forward primer and reverse primer. Since they are used in the same PCR reaction, it shall be ensured that the PCR condition is suitable for both of them.
One critical feature is their annealing temperatures, which shall be compatible with each other. The maximum difference allowed is 3 C. The closer their Tanneal are, the better.

17. Summary ~ when is a “primer” a primer?5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’

18. Summary ~ Primer Design Criteria
Uniqueness: ensure correct priming site;
Length: bases.This range varies;
Base composition: average (G+C) content around 50-60%; avoid long (A+T) and (G+C) rich region if possible;
Optimize base pairing: it’s critical that the stability at 5’ end be high and the stability at 3’ end be relatively low to minimize false priming.
Melting Tm between C are preferred;
Assure that primers at a set have annealing Tm within 2 – 3 C of each other.
Minimize internal secondary structure: hairpins and dimmers shall be avoided.

19. Diagram for PCR Primer Design
Sequence from which to choose primers
Primer Design
Results of search, including suggested annealing temperatures shown in list
PCR reaction parameters
Primer Selection Rules
Primer design is an art when done by human beings, and a far better done by machines. 

20. Web sites related to primer design
PCR primer designer: Primer3
Single sequence input:
Others: Oligo, BioTools, GCG, GeneFisher, Primer!, Web Primer, NBI oligo program, etc
Lists of other PCR software
Input protein alignment:
Input nucleotide sequences or nucleotide alignment:
List of primer design sites:
Melting temperature calculation software:
BioMath:

21. Common problems in primer design
self complementarity
upper-lower primer complementarity
excessive thermal stability
computer aided primer design

22. Hairpin formation

23. Primer dimer formation

24. Internal stability of the primers
Primers with stable 5’ termini give the best performance: reduces false priming on unknown targets
Low 3’ stability prevents formation of duplexes that may initiate DNA synthesis: 5’ end must also pair in order to form a stable duplex
Optimal terminal ∆G ~ 8.5 kcal/mol; excessive low ∆G reduces priming efficiency

26. Common problems in PCR Pre-PCR mispriming
Non-template nucleotide addition “Plus A artifacts”

27. Pre-PCR mispriming
undesired annealing between primers themselves or with ssDNA molecules at low temperature
DNA polymerase extends the misannealed primers, adding nucleotides to their 3’ end, rendering these modified primers unable to anneal to their intended sites and resulting in amplification of unwanted DNA

28. Solving Pre-PCR mispriming
Performing “HOT START PCR”
Reaction mix minus Taq
Template
Primers
dNTPs
Transfer to thermocycler
100 oC denaturation
80 oC hold
Add Taq polymerase
Start thermocycler

29. Degenerate Primers
Used to isolate novel genes on the basis of similarity and/or amino acid sequences
To amplify (fish out) conserved sequences of a gene or genes from the genome of an organism.
Used when attempting to design “universal” primers or when no unique area is found among aligned sequences

30. Nucleotide Represented
Degenerate Primers
Abbreviation Letter
Nucleotide Represented A  A C  C G  G T  T R
 AG Y
 CT M
 AC K
 GT W
 AT S
 CG B
 CGT D
 AGT H
 ACT V
 ACG N
 ACGT
eg:
5’-TCG AAT TCI CCY AAY TGR CCN T-3’
Y = pYrimidines = C / T (degeneracy = 2X) R = puRines = A / G (degeneracy = 2X) I  = Inosine  = C / G / A / T  N = Nucleotide = C / G / A / T (degeneracy = 4X)  

31. Degenerate Primers A potential degenerate primer:
ATG A[T/C]A AA[T/C] ATC CGA AAA AC[T/A/C]C A H Y Y

32. Degenerate Primers
Use greater primer concentration (>50 pmol) to compensate for degeneracy.
Competitive inhibition can be a problem
Multiple site annealing
No products formed

33. Universal Primers
Primers can also be designed to amplify multiple products. We call such primers “universal primers”. For example, design primers to amplify all HPV genes.
Strategy:
Align groups of sequences you want to amplify.
Find the most conservative regions at 5’ end and at 3’ end.
Design forward primer at the 5’ conservative region.
Design reverse primer at the 3’ conservative regions.
Matching forward and reverse primers to find the best pair.
Ensure uniqueness in all template sequences.
Ensure uniqueness in possible contaminant sources.

34. Semi-Universal Primers
Primers can be designed to amplify only a subset of template sequences from a large group of similar sequences. For example, design primer to amplify HPV type 1 and type 6 gene, but not other types.
Strategy:
Align all types of HPV genes.
Identify a subset of genes that are more similar to each other than to other subsets. In this case, type 1 and type 6.
Find the 5’ and 3’ regions that are conserved between type 1 and type 6, but are variable in other types.
Design forward primers from the 5’ region and reverse primers from the 3’ region.
Matching forward and reverse primers to find the best pair.
Ensure uniqueness in all template sequences.
Ensure uniqueness in possible contaminant sources.

35. Multiplex PCR
Multiple primer pairs can be added in the same tube to do the PCR
Good for amplifying multiple sites
Application example: genome identification
Design difficulty
Melting temperatures should be similar
No dimer formulation

36. Primer Design – Example
Design a pair of primers for sequence “NM_203378” in NCBI GenBank, so that the coding sequence of human myoglobin will be amplified using PCR reaction.
Between

37. Primer3

38. Primer3

39. Primer3