PCR Primer Design and Marker Development
Contact: Marwa Mahmoud Ghonaim marwa_ghonaim2006@yahoo.com
What is a primer? A primer is short oligonucleotides using in polymerase chain reaction to begin the amplification of the target region of DNA (random or specific regions) What is a primer design?
The creation of short nucleotide sequences for use in amplifying region of DNA.
Polymerase Chain Reaction Polymerase Chain Reaction (PCR) is the most common and often indispensable technique used in molecular biological research labs for a variety of applications. Amplifies a single or a few copies of DNA molecules to generate up to billion copies of a particular DNA sequence.
PCR primers are designed to: Highly conserved DNA regions Protein-coding regions More conserved regions that flank variable regions
The polymerase chain reaction (PCR) is a method by which DNA is amplified Method for exponential amplification of DNA sequences Sequences Basic requirements: –Template: DNA or RNA; –One or two DNA-oligonucleotides (also called DNA primers), are complementary to either end of the target sequence but lie on opposite strands; –Thermostable DNA polymerase (Taq, Tth, Pfupolymerases); –Desoxynucleotides (dATP, dTTP, dCTP, dGTP) and appropriate reaction buffer;
Temperature cycling 3 step process: • Denaturation temperature (usually 94-98°C) separates complemented DNA strands • Annealing temperature (usually 37-68°C) – primers hybridize to template • Extension temperature (usually 68-72°C) DNA-polymerase synthesized new DNA chain from primer
• Process repeated for approximately 20 to 40 cycles
The DNA synthesis and exonuclease activities of DNA polymerases
Why are primers important?
• Primers are what gives PCR its SPECIFICITY! • Good primer design
PCR works correctly
• Bad primer design
PCR works not efficient
PCR Primer Design
Primer design is a critical step in all types of PCR methods to ensure specific and efficient amplification of target sequence so it is the key for successful PCR.
General primer design guidelines Primer Specificity primers must be specific for desired sequence (conserved nucleotide or protein regions) to be amplified. Primer length primers should be long enough to ensure specificity (usually 18-30 bases), If the length is too short, it is difficult to design gene-specific primers and choose optimal annealing temperature;
Base composition • G+C content should be between 40% and 80%. Good primer must be: •no complementarity between forward and reverse primers or primers and product. •Melting temperature (55-80°C).
PCR primers design factors Characteristics of primers:
Thoughts on primer design:
Specificity Specific for the intended target sequence (avoid nonspecific hybridization)
Uniqueness Linguistic Complexity Length
Stability Forms table duplex with template under PCR conditions
Melting Temperature Annealing Temperature Stability at the 3′ end in primer
Compatibility Primers used as a pairs shall work under the same PCR conditions
Primer Pair Matching
Sequence linguistic complexity (LC) linguistic complexity : nucleotides arrangement and composition Linguistic complexity (LC) values for sequence length (s) are converted to percentages, in which 100% means maximal ‘vocabulary richness’ of a sequence: Primer sequence
LC, %
5’-AAAAAAAAAAAAAAAAAAAAA
8
5’-ACACACACACACACACACACA
15
5’-TTTTTTTTTTGGGGGGGGGAG
36
5’-GCTACCAATGAGAAGGTCACGT
98
5’-TGTTCTCCCATAGCACAAGAGGA
98
5’-TGGCTATTCTGAACCAGCGTTGC
100
Uniqueness The specificity of the oligonucleotides is one of the most important factors for good PCR; optimal primers should hybridize only to the target sequence, particularly when complex genomic DNA is used as the template. Amplification problems can arise due to primers annealing to repetitious sequences (retrotransposons, DNA transposons, or tandem repeats).
To improve Uniquenes
avoid regions of homology. Primers designed for a sequence must not amplify other genes in the mixture. You can BLAST the templates against the appropriate database. It will identify regions significant cross homologies in each template and avoid them during primer search.
Length Primer length has effects on uniqueness and melting/annealing temperature: • the longer the primer, the more chance that it’s unique • the longer the primer, the higher melting/annealing temp. (18-30 bases)
the longer the primer, the higher melting/annealing temp.
Melting temperature (Tm) calculation the temperature in degress Celsius, at which 50% of all molecules of a given DNA sequence are hybridized into a double strand, and 50% are present as single strands. The Tm is affected by a number of factors: Concentration of DNA. Concentration of ions in the solution, most notably Mg+ and K+. DNA sequence. Length of DNA.
Generally, sequences with higher fraction of GC base pairs, have a higher Tm than do AT-rich sequences. In a typical melting curve, you measure the increase in UV absorbance as the temperature increases.
The stability of the DNA double helix depends on a fine balance of interactions including hydrogen bonds between bases.
Annealing Temperature (Ta) calculation
The range of temperatures where efficiency of PCR amplification is maximal without non-specific products.
Generally, you should use an annealing temperature about 5°C below the Tm of your primers. The optimal annealing temperature (Ta Opt) for any given primer pair on a particular target can be calculated as follows:
Secondary Structure The
Presence of the primer secondary structures produced by
intermolecular or intramolecular interactions can lead to poor or no yield of the product. They adversely affect primer template annealing and thus the amplification.
Hairpins: It is formed by intramolecular interaction within the primer and should be avoided
Self Dimer: A primer self-dimer is formed by intermolecular interactions between the two (same sense) primers, where the primer is homologous to itself. Generally a large amount of primers are used in PCR compared to the amount of target gene. When primers form intermolecular dimers much more readily than hybridizing to target DNA, they reduce the product yield.
Cross Dimer: Primer cross dimers are formed by intermolecular interaction between sense and antisense primers, where they are homologous.
Intra-molecular interactions will give rise to hairpins
inter-molecular hybridization will give rise to dimers.
Primer dimer detection criteria
(A–C) Interactions between primers; (D) Hairpin structures; (E) Undesirable binding of primers to template sequence.
Primer-dimers involving one or two sequences may occur in a PCR reaction.
Stable primer dimer formation is very effective at inhibiting PCR since the dimers formed are amplified efficiently and compete with the intended target.
What is a primer-dimer
3’-end dimer:
5’-end and internal dimers:
Complementarity • PRIMER-PRIMER – Excessive similarity between primers, especially at the 3’ ends, leads to the formation of “primer dimers”
• PRIMER-TARGET – Ideally should be 100% similar for maximal specificity. – Primers don’t HAVE to be perfectly similar to target to work.
Web sites related to PCR primer designer PrimerDigital online tools: http://primerdigital.com/tools/ NCBI/Primer-BLAST (Primer3): http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Oligomer online PCR tools: http://www.oligomer.fi/en/analyysityokalut IDT online SciTools: http://idtdna.com/scitools/scitools.aspx MWG /Operon tools: http://www.eurofinsgenomics.eu/
sequence
PrimerID
Sequence(5'-3')
1F33_1_651-671
tacctctggggagcaacttgg
Tm(°C) ٥٩.٢
Primer_Quality(%) ٩٢
PCR_Fragment_Size(bp)
1R83_1_756-777
gtagctgatgaactcggagtgc
٥٧.٥
٩٥
1F39_1_745-764
ctgatcaagaagcactccga
٥٤.٧
٩٣
1R35_1_1788-1807
ccttgatgaccttgcagagg
٥٥.٨
٩٣
1F39_1_745-764
ctgatcaagaagcactccga
٥٤.٧
٩٣
1R27_1_1938-1957
acccagacatgctggagtcc
٥٩.٥
٩٣
1F47_1_1013-1033
tctacaagagcttgaccaacg
٥٤.٩
٩٣
1R73_1_1046-1065
gaagtgcttcacggcaagat
٥٦.٢
٩٣
1F47_1_1013-1033
tctacaagagcttgaccaacg
٥٤.٩
٩٣
1R35_1_1788-1807
ccttgatgaccttgcagagg
٥٥.٨
٩٣
1F47_1_1013-1033
tctacaagagcttgaccaacg
٥٤.٩
٩٣
1R27_1_1938-1957
acccagacatgctggagtcc
٥٩.٥
٩٣
1F48_1_1039-1058
gaggagcatcttgccgtgaa
٥٧.٩
٩٣
1R54_1_1419-1438
tggagtcctcgtggatgcca
٦١.٢
٩٣
1F48_1_1039-1058
gaggagcatcttgccgtgaa
٥٧.٩
٩٣
1R35_1_1788-1807
ccttgatgaccttgcagagg
٥٥.٨
٩٣
1F48_1_1039-1058
gaggagcatcttgccgtgaa
٥٧.٩
٩٣
1R30_1_1893-1912
gctccatgttggcggtccaa
٦١.١
٩٣
1F48_1_1039-1058
gaggagcatcttgccgtgaa
٥٧.٩
٩٣
1R27_1_1938-1957
acccagacatgctggagtcc
٥٩.٥
٩٣
Topt(°C) ١٢٧
٦٢
١٠٦٣
٦١
١٢١٣
٦١
٥٣
٥٨
٧٩٥
٦١
٩٤٥
٦١
٤٠٠
٦٣
٧٦٩
٦٢
٨٧٤
٦٤
٩١٩
٦٤
Primer Evaluation Primer list analysis
Name
Sequence
nt A T C G GC% Tm°C Molecular Weight(g/mole) nmol µg/OD260 Linguistic_Complexity(%) Primer's_PCR_Efficiency(%)
1f33_1_651-671
tacctctggggagcaacttgg
٢١ ٤ ٥ ٥ ٧
٥٧.١
٥٩.٢
٦٤٦٢.٢
٥.١
٣٢.٧
٨٧
٩٢
1r83_1_756-777
gtagctgatgaactcggagtgc
٢٢ ٥ ٥ ٤ ٨
٥٤.٥
٥٧.٥
٦٨١٥.٥
٤.٦
٣١.٦
٩٠
٩٥
1f39_1_745-764
ctgatcaagaagcactccga
٢٠ ٧ ٣ ٦ ٤
٥٠
٥٤.٧
٦٠٩٥
٥.١
٣٠.٩
٨٦
٩٣
1r35_1_1788-1807 ccttgatgaccttgcagagg
٢٠ ٤ ٥ ٥ ٦
٥٥
٥٥.٨
٦١٣٣
٥.٣
٣٢.٦
٧٦
٩٣
1f39_1_745-764
ctgatcaagaagcactccga
٢٠ ٧ ٣ ٦ ٤
٥٠
٥٤.٧
٦٠٩٥
٥.١
٣٠.٩
٨٦
٩٣
1r27_1_1938-1957 acccagacatgctggagtcc
٢٠ ٥ ٣ ٧ ٥
٦٠
٥٩.٥
٦٠٨٧
٥.٢
٣١.٩
٨٩
٩٣
1f47_1_1013-1033 tctacaagagcttgaccaacg
٢١ ٧ ٤ ٦ ٤
٤٧.٦
٥٤.٩
٦٣٩٩.٢
٤.٩
٣١.٣
٨٩
٩٣
1r73_1_1046-1065 gaagtgcttcacggcaagat
٢٠ ٦ ٤ ٤ ٦
٥٠
٥٦.٢
٦١٦٦.١
٥.١
٣١.١
٩٢
٩٣
1f47_1_1013-1033 tctacaagagcttgaccaacg
٢١ ٧ ٤ ٦ ٤
٤٧.٦
٥٤.٩
٦٣٩٩.٢
٤.٩
٣١.٣
٨٩
٩٣
1r35_1_1788-1807 ccttgatgaccttgcagagg
٢٠ ٤ ٥ ٥ ٦
٥٥
٥٥.٨
٦١٣٣
٥.٣
٣٢.٦
٧٦
٩٣
1f47_1_1013-1033 tctacaagagcttgaccaacg
٢١ ٧ ٤ ٦ ٤
٤٧.٦
٥٤.٩
٦٣٩٩.٢
٤.٩
٣١.٣
٨٩
٩٣
1r27_1_1938-1957 acccagacatgctggagtcc
٢٠ ٥ ٣ ٧ ٥
٦٠
٥٩.٥
٦٠٨٧
٥.٢
٣١.٩
٨٩
٩٣
1f48_1_1039-1058 gaggagcatcttgccgtgaa
٢٠ ٥ ٤ ٤ ٧
٥٥
٥٧.٩
٦١٨٢.١
٥.١
٣١.٣
٩٢
٩٣
1r54_1_1419-1438 tggagtcctcgtggatgcca
٢٠ ٣ ٥ ٥ ٧
٦٠
٦١.٢
٦١٤٩
٥.٣
٣٢.٦
٨٤
٩٣
1f48_1_1039-1058 gaggagcatcttgccgtgaa
٢٠ ٥ ٤ ٤ ٧
٥٥
٥٧.٩
٦١٨٢.١
٥.١
٣١.٣
٩٢
٩٣
1r35_1_1788-1807 ccttgatgaccttgcagagg
٢٠ ٤ ٥ ٥ ٦
٥٥
٥٥.٨
٦١٣٣
٥.٣
٣٢.٦
٧٦
٩٣
1f48_1_1039-1058 gaggagcatcttgccgtgaa
٢٠ ٥ ٤ ٤ ٧
٥٥
٥٧.٩
٦١٨٢.١
٥.١
٣١.٣
٩٢
٩٣
1r30_1_1893-1912 gctccatgttggcggtccaa
٢٠ ٣ ٥ ٦ ٦
٦٠
٦١.١
٦١٠٩
٥.٤
٣٣.١
٨٤
٩٣
1f48_1_1039-1058 gaggagcatcttgccgtgaa
٢٠ ٥ ٤ ٤ ٧
٥٥
٥٧.٩
٦١٨٢.١
٥.١
٣١.٣
٩٢
٩٣
1r27_1_1938-1957 acccagacatgctggagtcc
٢٠ ٥ ٣ ٧ ٥
٦٠
٥٩.٥
٦٠٨٧
٥.٢
٣١.٩
٨٩
٩٣
Primer Evaluation • Let’s assume we selected the first TCATTGTTTGCCTCCCTGC TAGAAACCCCAACCCGTGAAA primer pair (for + rev) • Website for online primer evaluation:
Enter Sequence
How we use primers in Retro-markers
There are several techniques using retroelements as molecular markers:
S-SAP (Sequence-Specific Amplified Polymorphism) IRAP (Inter Retrotransposon Amplified Polymorphism) REMAP (REtrotransposon Microsatellite Amplification polymorphisms) RBIP (Retrotransposons-Based Insertion Polymorphism) iPBS (inter Primer Binding Sites amplification)
SSAP (Sequence Specific Amplified Polymorphism) measure the distance from the transposon to the restriction site. Amplification is carried out between primers matching an LTR and a restriction site adapter ligated to genomic DNA digested with a restriction enzyme.
Inter-Retrotransposons Amplified Polymorphisms (IRAPs). IRAP product measures the distance between retrotransposon and another.
REMAP (Retrotransposon-microsatellite amplified polymorphism). Amplification is carried out between primers matching an LTR and a microsatellite domain (SSRs).
Retrotransposon based insertion polymorphism (RBIP). This technique can detect the presence and absence of retrotransposon.
The inter PBS amplification (iPBS) scheme
LTR retrotransposon structure: LTR and PBS sequence Two nested LTR retrotransposons in inverted orientations amplified from single primer or two different primers from primer binding sites. PCR product contains both LTRs and PBS sequences as PCR primers in the termini. In figure schematically showing general structure for PBS and LTR sequences, between 5’LTR(5’-..CA) and PBS (5’-TGG..3’) is spacer with several nucleotides (0-5 bases).