SEQUENCING PROBLEMS

Robert Chadwick rchadwick at nce-mail.com
Tue Nov 28 14:04:30 EST 2000


FYI
See the following web site for PCR sequencing tips.
http://gsu.med.ohio-state.edu/PCRRecommend.html

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 GoldTM
(Perkin-Elmer, Norwalk, CT), Platinum Taq (Gibco BRL, Gaithersburg, MD) and
TaqStartTM 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 MacVectorTM
(Oxford Molecular Group Inc., Oxford, United Kingdom), OligoTM (National
Biosciences Inc., Plymouth, MN), LaserGeneTM (DNASTAR Inc., Madison, WI) and
Primer ExpressTM (PE Applied Biosystems Inc., Foster City, CA). There are
also WWW sites that will assist in the design of primers. The web address
for one useful site is <http://www.williamstone.com/primers/index.html>
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 Tm) 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 (Perkin-Elmer,
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 PicoGreenTM 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	



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