GENESCAN TROUBLESHOOTING

GENESCAN TROUBLESHOOTING

GENOMICS
DNA Sequencing
Genotyping / GeneScan
Real-Time PCR
Oligo Synthesis
GeneChip Probe Array
Custom Spotted Array

PROTEOMICS
Protein Sequencing
Mass Spectrometry

BIOINFORMATICS
GCG / SeqWeb
Molecular Modeling
Internet Resources

Problems With PCR

Observation	Possible Causes	Recommended Actions
Faint or no signal from sample DNA	Sample contains PCR inhibitor (i.e.: heme compounds, EDTA, or certain dyes)	Quantitate DNA. Dilute if possible in order to ad minimum necessary volume. Repeat amplification.
		Wash the sample in an Amicon Centricon-100 column and repeat amplification. Note: For fragments smaller than 130 bp, use the Amicon Centricon-30 column instead.
		Add bovine serum albumin (BSA) to the PCR reaction mixture. (Use 8-16 µg BSA for every 50 µL PCR reaction volume.)
	Sample DNA is degraded	Evaluate the quality and concentration of the DNA sample by: Using the QuantiBlot Human DNA Quantitation Kit (for human DNA) Running an agarose yield gel If DNA is degraded or inaccurately quantitated, re-amplify with an increased amount of DNA.
	Insufficient sample DNA added because of inaccurate quantitation	Evaluate the quality and concentration of the DNA sample by: Using the QuantiBlot Human DNA Quantitation Kit (for human DNA) Running an agarose yield gel If DNA is degraded or inaccurately quantitated, re-amplify with an increased amount of DNA.
	Incorrect pH	Verify buffer pH and concentration. If incorrect, quanititate sample DNA. Too little or too much DNA can alter the pH.
	Primer choice not optimal (i.e.: primers may be annealing to sites of template secondary structure or may have internal secondary structure)	Use different primers.
	T_m of primers is lower than expected	Decrease the annealing temperature by 2ƒC increments.
Poor yield for multiplex PCR	Non-optimal thermal cycling parameters	Between the denaturation and annealing stages, add a 2 minute down-ramp time to thermal cycling profile. Note: For multiplex PCR, a short down-ramp time is not necessarily optimal.
Poor yield for multiplex PCR	Competition from mispriming and other competing side reactions	Use AmpliTaq Gold DNA Polymerase.
Yield gets progressively poorer for successive PCR amplifications performed over time	Expired or mishandled reagents	Check expiration dates on all reagents. If not expired, verify that reagents are being stored and used according to manufacturer’s instructions. Compare with PCR performance using fresh reagents.
Inconsistent yields with control DNA	Combined reagents not spun to bottom of PCR sample tube	Place all reagents in apex or tube and spin briefly after combining.
	Combined reagents left at room temperature or on ice for extended periods of time (encouraging mispriming and other primer artifacts)	Put tubes in block immediately after combining reagents.
	Combined reagents not thoroughly mixed	Vortex all primers, reagents, and reaction mixtures (minus enzyme) thoroughly to ensure uniform concentration.
	Primers not uniformly suspended before adding to reaction mixture. (Primers can aggregate and settle to the bottom of the tube.)

Problems with Extra Peaks

Observation	Possible Causes	Recommended Actions
Extra peaks appear with no discernible pattern	Presence of exogenous DNA	Use appropriate techniques to avoid introducing foreign DNA during laboratory handling.
	Non-specific priming (i.e.: primer-template mismatch)	Check for good primer design.
		Add less template DNA Note: High DNA concentrations promote non-specific annealing
		Optimize Mg²⁺ concentration.
		Add less primer DNA Note: High primer concentrations promote non-specific annealing.
		If you are not using AmpliTaq Gold DNA Polymerase, consider using a Hot-Start Technique.
		Increase annealing temperature in 2-5ƒC increments.
		Decrease annealing and/or extension times.
		Increase primer length.
		Perform a second amplification with nested primer.
		Perform Touchdown PCR.
	Primer-dimer and primer-oligomer artifacts (likelihood increases with multiplex PCR)	Check primers for 3’ complementarity.
		Design longer primers.
		Reduce primer concentration.
		Reduce number of cycles.
		Raise the annealing temperature in 2-5ƒC increments.
		Increase amount of target DNA.
	Incomplete restriction (and/or ligation if performing AFLP)	Repeat restriction (and /or ligation)
	If performing AFLP, too much DNA in reaction so that insufficient adaptor is present	Use the recommended amount of template DNA.
	Mixed sample	Verify quality and integrity of sample.
Presence of split peaks differing in size by one base pair (Extra peak of size n+1)	Partial non-template addition of an extra nucleotide (usually adenosine) to the blunt end of the PCR product	Add the correct amount of Mg²⁺ to the reaction mix. Note: Increasing the Mg²⁺ concentrations can increase the frequency of non-templated nucleotide addition and vice versa. Increasing the extension time at 72ƒC will increase the frequency of non-template nucleotide addition.
Presence of peaks differing in size by two, three, or four base pairs (Extra peaks of size n-2, n-3, or n-4)	Stutter product formed during amplification of di-, tri-, or tetranucleotide STR loci	See "Stutter Products"

Problems With Missing Peaks

Observation	Possible Causes	Recommended Actions
Some but not all loci visible on electopherogram	Sample DNA is degraded (indicated if shorter amplicons are favored)	Quantitate DNA and add more template. Repeat amplification.
		Wash the sample in an Amicon Centricon-100 column and repeat amplification. Note: For fragments smaller than 130 bp, the Amicon Centricon-30 column is preferable.
	Sample contains PCR inhibitor (i.e.: heme compounds, EDTA, or certain dyes)	Quantitate DNA and add minumum necessary colume of PCR product. Repeat amplification.
Individual alleles are missing when inheritance data is examined	Mutation in primer annealing site of one allele	Change the primer.

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