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General Recommendations

The most important factor in obtaining high quality sequence data is that your PCR amplification must be clean, robust and specific. One of the best methods of improving PCR specificity and reliability is to use a hot start PCR. This prevents the PCR reaction from beginning until elevated temperatures are reached and prevents mispriming due to potential regions of close homology in the genomic DNA. Primer-dimers due to regions of complementary base pairing in the amplification primers are also largely prevented from forming in a hot start PCR reaction. There are three methods of doing hot start PCR:

DNA Polymerase enzyme or another critical reagent is not added until the reactions reach approximately 80°C.

The enzyme is separated from the primers or magnesium by using a wax bead overlay that melts at a higher temperature.

A hot start DNA Polymerase is used which is inactive until it reaches elevated temperatures.

The latter is the easiest way to do a hot start. AmpliTaq Gold^TM (Applied Biosystems, Norwalk, CT), Platinum Taq (Gibco BRL, Gaithersburg, MD) and TaqStart^TM Antibody (Clontech, Palo Alto, CA) are examples of heat-activated thermostable DNA polymerases.

Another useful tool in obtaining good PCR and sequencing results is the use of software for PCR primer design. There are several software packages that assist in the selection of PCR primers. Examples of software which assist in the selection and design of PCR and sequencing primers are MacVector^TM (Oxford Molecular Group Inc., Oxford, United Kingdom), Oligo^TM (National Biosciences Inc., Plymouth, MN), LaserGene^TM (DNASTAR Inc., Madison, WI) and Primer Express^TM (Applied Biosystems Inc., Foster City, CA). There are also WWW sites that will assist in the design of primers. Click here to see our Primer Design Workshop Presentation.

Most of these DNA analysis software packages calculate the melting temperatures of the primers, determine if the primers have regions of complementary base pairing, calculate the expected size of PCR products, and determine the optimal annealing temperature to use in the thermal cycling. Primers with melting temperatures that are within a few degrees of 60°C are recommended. Typically if a primer is 20 or more bases in length and is about 50% Gs and Cs, the melting temperature will be near 60°C. Primers that have single base repeat regions of more that four bases should not be selected. Avoid primers that have regions of potential complementary base pairing, particularly at the 3' ends of the primers. Also, complementary base pairing within a primer can cause hairpins to form and this should be avoided with proper primer design. Melting temperatures (or T_m) for primer pairs should be within 3°C of each other. The nearest neighbor method is considered to be the most accurate mathematical method of determining melting temperatures.

Using low PCR primer and dNTP concentrations in the PCR reactions can improve both amplification and sequencing results. Lower PCR primer concentrations help to reduce primer-dimer artifacts and interference in sequencing reactions from potential PCR primer carry over. Lower dNTP concentrations can help to prevent sequencing problems due to altered d/ddNTP ratios from PCR dNTP carryover. PCR reaction primer concentrations of less than 200 nM and dNTP concentrations of less than 100 mM typically give good results.

One method that can greatly simplify subsequent sequencing steps is to amplify the region of interest using PCR primers with "M13 tails". This creates universal priming sites and allows the use of the same sequencing primers for all the PCR amplification products, even those of different genomic regions. Since the same sequencing primers are used for all the templates, optimization of the sequencing reactions has already been done. However, this technique requires that amplification primers be synthesized with the -21M13 sequence (TGT AAA ACG ACG GCC AGT) on the 5' end of one PCR amplification primer and the M13 Reverse sequence (CAG GAA ACA GCT ATG ACC) on the 5' end of the other PCR amplification primer. Since these different universal priming sites are created in the subsequent amplifications, both strands of all the PCR products can be readily sequenced using two template/primer mixes containing either the forward or reverse sequencing primers.

Sequencing of both strands of a double stranded PCR product is very useful for confirmation of suspected mutations. Alignment of both sequence directions nearly always resolves artifacts such as G/C compressions or other anomalous mobilities due to DNA secondary structure. This greatly helps to eliminate both false positive and false negative mutation detection rates.

PCR Conditions

Recommended amplification conditions to try initially are given for a 25 mL PCR reaction volume. Concentrations of the following reagents typically give good results: 100 nM of each of the respective PCR primers, 25 ng of genomic DNA, 100 mM of each dNTP, 1.0 U Taq Gold DNA Polymerase (Applied Biosystems, Norwalk, CT), 10 mM pH 8.3 Tris-HCL, 50 mM KCl, and 2 mM MgCl₂.

Following is a recommended cycling profile to try initially when amplifying with "tailed" primers:

Initial denature and Taq Gold activation

1 cycle of

95°C 10 minutes

Followed by

8 cycles of

98°C 10 seconds

60°C 30 seconds

70°C 1 minute

Followed by

32 cycles of

96°C 10 seconds

68°C 1 minute

Followed by

Soak 4°C forever

The first eight cycles are done using three step cycling since only the 3' ends of the "tailed" primers are initially annealing to the genomic target. Later two step cycling is done since the entire tailed primer is annealing primarily to the newly synthesized template copies with the newly created M13 priming sites. Since the tailed primers are typically about 38 bp long, they have high melting temperatures and a two step anneal/extend cycling profile can be used in the later cycles.

QC and Yield Determination

Amplification success is determined by the standard agarose gel method. Load 10 mL of the PCR reactions on a 1% Seakem GTG agarose gel containing ethidium bromide in the gel solution at a concentration of 0.8 mg/mL. Molecular weight standards of known concentration and size should also be loaded and electrophoresed simultaneously. Amplifications should give a single band of the expected size. If more than one band is seen the PCR products will not sequence well without further optimization. However, if you see a band of the proper size on the agarose gel, there should be enough PCR product to cycle sequence.

Amplification yields can also be determined very accurately by spectroflourimetry using a dye which selectively stains double stranded DNA such as PicoGreen^TM dsDNA quantitation reagent (Molecular Probes, Inc., Eugene, OR).

Design of Sequencing Primers

If "tailed" PCR primers are not used, custom sequencing primers or one of the PCR amplification primers must be used as a sequencing primer. Note that the sequence obtained immediately following the primer is usually not readable for at least 20 bases 3' of the end of the sequencing primer. The length of high quality sequence data that can be reliably detected is typically approximately 500 bases. Often longer reads than this are possible, but peak broadening makes the determination of heterozygotes less reliable after 500 bases. As stated previously, an advantage of amplifying with "tailed" PCR primers is that the same sequencing primers are used to sequence all templates and optimization of the sequencing reactions has already been done.

Purification of PCR Products

The most common methods to purify PCR products are by enzymatic treatment, spin column chromatography, and gel purification. Enzymatic purification is detailed below. For column chromatography purification, the QiaQuick PCR purification kit (QIAGEN, Chatsworth, CA) or Microcon-100 (Millipore, Bedford, MA) is recommended. Gel purification of PCR products involves cutting the PCR product of interest out of an agarose or acrylamide gel. If agarose gels are used, enzymatic digestion of the agarose with agarase (Sigma Chemicals, St. Louis, MO) is often performed. If acrylamide gel purification is used, purification by either elution or electrophoresis of the excised band are common techniques. However, since gel purification involves exposing the PCR products to UV light which can nick or crosslink the DNA, the Exo1/SAP enzymatic digestion or column purification methods are preferable and recommended.

The simplest method of PCR purification is by enzymatic digestion of the oligonucleotide primers and deoxynucleotides. Following is the protocol for exonuclease 1/shrimp alkaline phosphatase enzymatic purification:

Aliquot 10 mLs of PCR product to a microamp tube and place the tube on ice.

Add 1 mL Exonuclease 1 (10 Units/mL, Amersham Life Science) to the aliquoted PCR product.

Add 1 mL Shrimp Alkaline Phosphatase (2 Units/mL, Amersham Life Science).

Mix well by pipetting repeatedly.

Incubate at 37°C for 15 minutes (NO longer!). It is easiest to do this in a thermal cycler since a thermal profile can be created that links this step and the following steps.

Incubate at 80°C for 15 minutes to kill the enzymes.

Store the products at 4°C until they are sequenced.

Note that the above purification method will not remove double stranded DNA contaminants, such as primer-dimers or non-specific amplicons. Spin column or gel purification can be effective in removing these types of contaminants.

Organization Name	Address	City	State	Postal Code	Phone Number	Fax Number
USB Specialty Biochemicals	26111 Miles Rd	Cleveland	OH	44128-	(800) 321-9322	(800) 535-0898

Product Name	Product Description	Price	Catalog Number	Unit
PCR Product Presequencing Kit	EXOl/SAP kit	60.00	US70995	ea