Dr marwa mahmoud ghonaim introduction in retro markers and primer design

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).