DNA Sequencing and Fragment Analysis

Posts tagged ‘Primers’

Guidelines for Optimizing PCR: Concentration of Target DNA and Primers

The Polymerase Chain Reaction, or PCR, is a basic method used in molecular biology to produce copies of a small target region of DNA in a sample. The basics to PCR were discussed previously here. The copies of DNA produced by PCR provide researchers with sufficient copies for other applications in research including automated Sanger sequencing. Although there is basic methodology to most PCR methods, each reaction is different and requires optimization, a process for adjusting variables and producing a single desired product.  There are several factors to consider when optimizing PCR such as total copies of target DNA, primer concentration, MgCl2 and deoxynucleotides, or dNTPs.  Some of these variables depend on the total volume of the PCR reaction because the final concentration of the components in PCR should be constant depending on whether the reaction is 25 ul, 50 ul or 100 ul. In this article we will focus on two variables, the number of copies of the target DNA and primer concentration.

The Template: Target DNA

Generating copies of a target DNA region using PCR applications is not as sensitive to the quality of the template DNA when compared to Sanger sequencing. However, it is still advisable to use a relatively pure DNA sample free from salts and other contaminants. Clean template DNA has a better probability to generate a clean PCR product. The final diluted sample of target DNA is better diluted in water rather than buffer because buffers can interfere with difficult PCR amplifications.

The most important aspect of the target DNA to consider is the total number of copies in the reaction available for amplification. The target DNA provides the initial template for the amplification of the first set of products amplified and continues to provide the template for the remaining cycles. As PCR products are generated, they also provide copies of the target DNA used as a template for amplification. This is what allows PCR to generate millions of copies of a target region. Therefore, it is important that sufficient copies of the original target DNA are present in the reaction. Too many copies of the original target can lead to generation of false products early in PCR that also act as template DNA. The template DNA isolated from bacteria may consist of only a 2 million-base genome whereas the human genome has 3 billion bases. Therefore, bacterial genomic DNA will have far more copies of the target in a 50 ng sample than human DNA. For bacterial DNA 10E5 copies will require only 300 picograms of DNA. For human DNA 10E5 will require over 300 nanograms of DNA, a one million fold difference.

PCR conditions generally recommend 10E4 to 10E5 copies of the target DNA in the reaction independent of the total volume. There is some flexibility in the copy number of the target sequence. However, more copies of the target DNA will reduce specificity of the PCR reaction and likely produce a greater number of false products. The total number of cycles for PCR should be reduced when higher concentrations of target DNA are in the reaction.

Concentration of the Primers

Primers are the determining factor of what region of the DNA will be amplified by PCR. The forward and reverse primer must have an exact base match with the beginning and end of the target region. Excessive primer concentration is perhaps one important factor that often causes generation of false products in a PCR. Too much primer reduces specificity and this will allow primers to anneal in regions of the template that are not the target region. The results of excessive primers are often seen in unclean Sanger sequencing results because false products can be sequenced along with the desired target. The amount of forward and reverse primer should be limited to reduce potential false priming. Excessive concentrations of forward and reverse primers can also cause formation of primer dimer when the primers anneal and amplify themselves independent of the target DNA.

Primer concentration is one variable dependent on the total volume of the PCR reaction in order that sufficient copies of the primer find the target annealing sites. A total concentration of 0.5 micro-Molar (uM) to 1 uM is generally sufficient to amplify most target regions, although a smaller concentration may also work in some applications. Typically our lab uses a final concentration of 0.8 uM for most PCR reactions. The final judgment on primer concentration will be viewed after products are electrophoresed on an agarose gel in order to show the number of products amplified.

We use a relatively simple calculation to dilute primers to a final concentration of 10 uM as shown starting with the primary primer concentration of 1 micro-grams (ug)/ micro-liter (ul). It requires that the molecular weight (MW) of the primer is known and should be provided along with the primer.

1 ug/ul  *1umol/MW (ug) *10E6 ul/l  = concentration umol/l which equals  uM

A primer with the final concentration of 200 uM will be diluted by adding 1 ul of the primer to 19 ul of water for a final concentration of 10 uM. This is our working concentration for PCR. For a final concentration of 0.8 uM, 2 ul of the forward and reverse primer are added to a 25 ul reaction whereas 8 ul of each would be added to a 100 ul PCR reaction.

Primer concentration is one of the more important variables to consider when optimizing a PCR reaction. Concentrations greater than 1 uM could often lead to primers annealing along non-target regions and the generation of false products.  Insufficient concentrations of either primer could result in little or no amplification.

Please go here if you would like to download a

reprint for this article in pdf format

Primer Selection Guidelines: Good Primers Important for PCR and Automated Sequencing

DNA synthesis is the production of short, single-stranded DNA molecules (called primers or oligonucleotides) often used in the polymerase chain reaction (PCR) and DNA amplification for Sanger sequencing applications. The region of DNA amplified is determined by an exact match of the primer to its complimentary bases on a given DNA strand. Primer sequences are determined from known sequence since there must be a match to the region of DNA to be amplified.

PCR amplification requires 2 primers that determine the region of sequence amplified in the forward and reverse direction. The forward primer is designed along one strand in the direction toward the reverse primer. Likewise, the reverse primer is designed from the complimentary strand. PCR is exponential amplification in which the newly generated PCR fragment from one cycle also acts as a template for the next cycle (Figure 1A).

Amplification of DNA for Sanger sequencing differs from PCR in that a single primer is used. Amplification is the reproduction of one strand using the compliment from the original strand. Newly generated PCR fragments are single stranded and do not provide a complimentary strand that could act as a template for additional amplification (Figure 1B).

Differences in amplification between PCR and Sanger sequencing were discussed previously (Sanger Sequencing Amplification Compared to Basic PCR).

Simply designing the sequence of the primer from known sequence does not ensure the primer will anneal to the desired region and initiate amplification. The primer should be designed following a set of given standards that improve the chances of success. The guidelines for designing primers used in PCR and sequencing are fairly similar. Primers are designed in the 5’ to 3’ direction to compliment the direction of amplification.

For those utilizing PCR and Sanger sequencing in everyday applications, primer design could seem like yesterday’s news. However, we still believe reviewing good primer design guidelines is helpful to any researcher involved in genetic research.

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.

Primer design is an important aspect relating to many forms of PCR including basic PCR, fragment analysis, quantitative analysis and Sanger sequencing. Below is a link that offers free primer design software…

http://frodo.wi.mit.edu/

We invite any additional guidelines or comments that may be useful to both experienced researchers and those new to primer design.

Please go here if you would like to download a

reprint for this article in pdf format