Guidelines for Primer DesignG1. Pri

Guidelines for Primer Design

G1. Primer length should be in the range of 18 to 22 bases. Primers less than 18 bases will have a low melting temperature (Tm values) and might not anneal to the template. There is some flexibility for designing primers longer than 18 bases. Longer primers are frequently designed from template regions that are AT-rich and need additional bases to increase the Tm value.

G2. The primer should have GC content of 50% to 55%. This is the equivalent of 9 or 10 GC bases included in an 18 base primer. Sometimes there are regions on a template that are AT-rich which prevents meeting this guideline. In those cases it is recommended to design a primer longer than 18 bases.

G3. Primers should have a GC-lock on the 3’ end. A GC-lock is designed when 2 of the final 3 bases is a G or a C. The 3’ base should always be a G or a C.

G4. The melting temperature of any good primer should be in the range of 50OC to 55OC. However, guidelines particularly related to Tm value have some flexibility. Melting temperatures are directly related to the PCR cycle annealing temperature. Tm values that are too low may not anneal well during PCR. High values could be too stringent causing difficulty locating the correct annealing site on the template.

G5. The primer should not include poly base regions. This is when 4 or more bases in a row are the same. This guideline helps prevent potential slippage in which the primer shifts from the annealed position.

G6. Four or more bases that compliment either direction of the primer should be avoided. This prevents the primer from annealing to itself and forming what is referred to as primer-dimer. Primer-dimers have the capability of amplifying the primer itself causing short secondary sequence.

PCR Specific Guidelines

G7. Forward and reverse primers used in PCR amplification should have similar melting temperatures (+/- 2OC). This allows a 4OC difference in total melting temperatures. Researchers involved in using PCR amplification will use primer Tm values in an effort to optimize PCR cycles. Similar Tm values for forward and reverse primers aid optimization efforts. Multiplex PCR applications using multiple primer pairs should all have similar Tm values. A wide range in primer melting temperature complicates PCR optimization.

G8. Forward and reverse primers should not have regions 4 bases or longer that compliment. Just like a primer used in Sanger sequencing, forward and reverse primers used in PCR can anneal to each other and form primer-dimers.

G9. The Tm values for tailed primers should include the tail in calculating melting temperature. Yes, melting temperatures will be greater than 55OC. However, the additional bases in the tail will add to the amplified PCR fragment and become part of the priming site. Tailed primers are often used to add restriction sites to an amplified product.

Results (Indonesian) 1:

Pedoman untuk desain PrimerG1. Primer panjang harus dalam kisaran basis 18-22. Primers kurang dari 18 basis akan memiliki temperatur leleh yang rendah (Tm nilai) dan mungkin tidak Anil ke template. Ada beberapa fleksibilitas untuk merancang primers lebih dari 18 basis. Lagi primers sering dirancang dari daerah template yang di-kaya dan perlu tambahan basis untuk meningkatkan nilai Tm.G2. Primer harus memiliki konten GC 50% untuk 55%. Ini adalah setara dengan 9 atau 10 GC basis termasuk dalam primer dasar 18. Kadang-kadang ada daerah pada template yang di-kaya yang mencegah pertemuan pedoman ini. Dalam kasus-kasus dianjurkan untuk merancang primer lebih dari 18 basis.G3. Primers harus memiliki GC-lock pada akhir 3'. GC-lock dirancang ketika 2 dari akhir dasar 3 G atau C. 3' dasar harus selalu G atau C.G4. Suhu mencair primer baik apapun harus berada dalam kisaran 50oC untuk 55OC. Namun, pedoman terutama yang berkaitan dengan nilai Tm memiliki beberapa fleksibilitas. Suhu mencair secara langsung berhubungan dengan siklus PCR Anil suhu. Nilai-nilai TM yang terlalu rendah mungkin tidak Anil baik selama PCR. Nilai-nilai yang tinggi bisa menjadi terlalu ketat menyebabkan kesulitan menemukan situs Anil yang benar pada template.G5. Primer tidak harus mencakup daerah basis Poli. Ini adalah ketika 4 atau lebih berturut-turut adalah sama. Pedoman ini membantu mencegah slip potensial di mana primer bergeser dari posisi annealed.G6. Four or more bases that compliment either direction of the primer should be avoided. This prevents the primer from annealing to itself and forming what is referred to as primer-dimer. Primer-dimers have the capability of amplifying the primer itself causing short secondary sequence.PCR Specific GuidelinesG7. Forward and reverse primers used in PCR amplification should have similar melting temperatures (+/- 2OC). This allows a 4OC difference in total melting temperatures. Researchers involved in using PCR amplification will use primer Tm values in an effort to optimize PCR cycles. Similar Tm values for forward and reverse primers aid optimization efforts. Multiplex PCR applications using multiple primer pairs should all have similar Tm values. A wide range in primer melting temperature complicates PCR optimization.G8. Forward and reverse primers should not have regions 4 bases or longer that compliment. Just like a primer used in Sanger sequencing, forward and reverse primers used in PCR can anneal to each other and form primer-dimers.G9. The Tm values for tailed primers should include the tail in calculating melting temperature. Yes, melting temperatures will be greater than 55OC. However, the additional bases in the tail will add to the amplified PCR fragment and become part of the priming site. Tailed primers are often used to add restriction sites to an amplified product.