5 % Stutter vs Peak Height At low concentrations stutter increases – a stochastic effect?Poor quality samples from casework may also cause problemsdue to inhibition.
6 Extract of Pistol Grip and Trigger Relatively low amount of amplified DNA?BlueGreenYellowEnhanced stutter or mixture?
7 Overloaded peaks will also show relatively high stutter Non specific amplification-AStutterTruncated peaks give wrong ratios for peak stutterWhy else is overloading bad?1. raised baseline2. non specific amplification3. peak height ratios4. -A
8 Degradation and PCR Inhibition Degradation affects larger alleles more, however there is no published study on the “threshold at which degradation is apparent”The amplification efficiency of each set of alleles varies independently and differential amplification across loci can occur – Moretti, JFS 2001Low quality formamide can mimic the degradation effectInhibition generally affects certain loci more than others and may or may not produce a slope effect- McCord, unpublishedThere are several likely mechanisms for inhibition including DNA aggregation, Protein-DNA binding, chelation of Mg, interference with primer binding, etc.
9 DNA Degradation Non-degraded Positive Control 20 pg/µL (0.250 ng/12.5 µL)Degraded Bone Sample20 pg/µL(0.250 ng/ 12.5 µL)
10 Degraded DNA and Amplification With degraded DNA two injections may be necessary to keep data on-scaleAllele drop-out-A peakOversaturationEnhanced stutterAllele drop-out“Pull up”
11 Non-DNA Contamination/Inhibition Anything that is water soluble may co-extract with DNA unless a capture technique is used.For capture techniques anything with a similar chemical property to DNA may co-extractDetergents, metal ions, humic substances are all potent contaminant/inhibitorsCan cause all sorts of strange effects includingSpikes, dye blobs, elevated baselines, loss of signal, odd current effects
12 Degraded and inhibited bone sample Loss of larger alleles due to degradationdifferential amplification at certain loci (inhibition?)Powerplex degraded sample bone sample found by creek (jane)
13 2. Sample Issues Formamide Conductivity Excessive salt in sample due to evaporationMetal ion contaminationSensitivity issues with Microcon cleanup (salt removal)Dye “blobs” – artifacts from primer synthesis
14 Attributed to poor formamide Golden Gate EffectAttributed to poor formamide
15 Sample Problem?. Check ROX, looks OK 320 V/cm 47 cm uncoated capillary POP4 Polymer
16 Answer: Incomplete denaturation of standard due to poor quality formamide320 V/cm 47 cm uncoated capillaryPOP4 Polymer
17 Post PCR manipulationReprocessing post PCR to concentrate samples can improve signal but be carefulPCR sample is concentrated but:Spin filtration may result in removal of background salts,This can greatly enhance sensitivity due to the stacking processBest idea- remake sample up in buffer, not water to avoid reading stochastic effects.
19 Dye Blobs and their Removal Residual dye artifacts439389II438437391389I426YCAII a/b390385 a/b393392H446019388448447100 bp139200250*300150160340350Dye blob removal with Edge columns6FAM(blue)391439389II389I437438385 a/bVIC(green)YCAII a/b390426393NED(Yellow)39246019H4388PET(Red)447448LIZ(Orange)139160100 bp350150200250*300340NIST Y-STR 20plex assayButler, J.M. (2005) Constructing STR multiplex assays. Methods in Molecular Biology: Forensic DNA Typing Protocols (Carracedo, A., ed.), Humana Press: Totowa, New Jersey, in press.
20 3. External Factors Room temperature Cleanliness Variations in room temperature can cause mobility shifts with band shifts and loss of calibrationTemperature is also important due to effects of high humidity on electrical conductanceCleanlinessUrea left in sample block can crystallize and catalyze further crystal formation causing spikes, clogs and other problems.Best bet is to keep polymer in system and not remove or change block until polymer is used up.
21 Temperature effects Viscosity – mobility shift ep = q/6rDiffusion – band broadeningDNAConformation – DNA size based sievingvs ep = q/6rCurrent – PowerP= VI = I2RIncreased current internal temperature rise diffusion band broadening
22 Likely the result of temperature or viscosity induced mobility change Band shift in the FGA locusLikely the result of temperature or viscosity induced mobility change
23 Effect of Temperature on allele size Slope is 0.14 bases/degree centigradeTherefore a small change in temperature has a big effect(A 1-2 degree shift in temperature of the heat plate can produce an OL allele)
24 Due to its structure and its non-calibration, the “250”peak can be used to indicate stabilityInitial RunStability losses toTemperatureElectroosmosisSyringe leaksAdsorptionExcess currentBlockages
32 Monitoring Room Temperature Over Time ± 10 oC spread (over many weeks)
33 Use of Second Allelic Ladder to Monitor Potential Match Criteria Problems 1st Injection (standard for typing)15th Injection (treated as a sample)-0.75 bp-0.54 bpThese alleles have drifted outside of their genotyping bins due to temperature shifting over the course of the sample batch
34 What to do if calibration is lost What to do if calibration is lost? The 310 only calibrates to the first run ladder this ladder sample may have been run at a different temperature!If protocol permitsGo to the next ladderRerun sampleCheck currentCheck allelic ladderAlways check the ROX size standardLook for extra bandsCheck peak heightCheck parameters and alignment
35 CleanlinessUrea sublimates and breaks down to ionic components - these find a path to groundSimilarly wet buffer under a vial creates paths to groundCapillary windows must be clear or matrix effects will occurLaser will often assist in this processVial caps will transfer low levels of DNA to capillary
36 Keep Your System Clean! Carbon Trails High Humidity or wet buffer vialscan create otherpaths to groundKeep Your System Clean!
37 4. Instrumental Factors Optical System Sensitivity changes with age, capillary diameter, capillary cleanliness, instrument calibrationFluidic SystemEffects of bubbles, dust, urea crystals, leaks in syringe and capillary ferruleMatrix CalculationsChanges in buffer, optics, sample dye can alter the software calibrationsCapillary ProblemsChemisorbed materials on capillary surface can produce osmotic flow, DNA bandbroadening and inconsistant resolution (meltdowns)
38 Consider the optical system Laser(488nm)Charged coupled deviceLaserCapillaryCCDOpticsGrating
39 Issues with the Optical System Pay attention to signal to noise, not absolute peak intensityArgon Ion lasers outgas and eventually loose intensity; take note of laser current and monitor it over timeFluorescence expression:If = I0kεbCφ - changes in input intensity, I0- changes in capillary diameter, b- cleanliness of capillary, kAll these things directly affect peak RFUs, however, baseline noise is more affected by detector.Thus by monitoring signal to noise, you can get a better picture of your optical system.
40 The Detection Window Capillary Detection Window Make sure that the capillary window is lined up (if it is not, then no peaks will be seen)Detection WindowWindow may need to be cleaned with ethanol or methanolReview Start of Raw Data CollectionCapillaryLittle spikes indicate need to change buffer… check current
42 Fluidic Problems Syringe leaks Viscosity changes At the barrelAt the capillary nutAt the capillary windowViscosity changesWater in the blockBubblesTemperatureCapillary conditioningPreelectrophoresisclogging
43 Buffer IssuesThe buffer and polymer affect the background fluorescence- affecting the matrixUrea crystals and dust may produce spikesHigh salt concentrations may produce reannealing of DNAHigh salt concentrations affect currentLow polymer concentrations affect peak resolution
44 Current SpikesGenerally appear in all lanes and are sharper than regular peaksThese are a natural consequence of the application of high voltage in CE
45 Remove all bubbles from the channels Bubbles in the channels can prevent flow of ions and are usually exhibited by zero current when the voltage is applied
47 These spikes resulted from buffer dilution with poor water These spikes resulted from buffer dilution with poor water. The problem disappeared when the HPLC grade water was purchased to dilute buffer and samplesSalt/ particulates problem (buffer issue causing spikes) Analyst used tap water to dilute problem dissappeared when switched to ultrapure dilution water.
48 Beware of Urea Crystals Urea crystals have formed due to a small leak where the capillary comes into the pump blockUrea sublimates and can evaporate to appear elsewhereUse a small balloon to better grip the ferrule and keep it tightPump block should be well cleaned to avoid problems with urea crystal formation
49 Storage when ABI 310 is not in use Keep inlet of capillary in water…if it dries out then urea crystals from the polymer will clog the openingThe waste vial (normally in position 3) can be moved into positionA special device can be purchased from Suppelco to rinse the capillary off-lineStore in distilled waterNote that the laser is on when the instrument is onRemember that the water in the open tube will evaporate over time…
50 Matrix ProblemsA poor matrix can lead to raised baseline and therefore calling of too many peaksLarger sized alleles will not be identified as peaks because the GeneScan table for a particular dye color has filled up
51 Note negative peaks and funny downward spiking baseline Effect of bad MatrixNote negative peaks and funny downward spiking baselineM2 ladder Matrix problem
52 Good Capillary in 3100 Array Capillary MeltdownsIdentifiler data(A) Good resolution(B) Poor resolution6FAM (blue)D8S1179D21S11D7S820CSF1POTH01VIC (green)D3S1358D13S317D16S539D2S1338NED (yellow)TPOXD19S433VWAD18S51PET (red)AMELD5S818FGAFigure 6. Comparison of same sample with good resolution (A) and poor resolution (B) due to a bad capillary. The STR kit used was the AmpFlSTR® Identifiler™ (5dye). Results from different capillaries on an ABI 3100 array.LIZ (orange)GS500 LIZ size standardBad Capillary in 3100 ArrayGood Capillary in 3100 ArrayButler, J.M., Buel, E., Crivellente, F., McCord, B.R. (2004) Forensic DNA typing by capillary electrophoresis: using the ABI Prism 310 and 3100 Genetic Analyzers for STR analysis. Electrophoresis, 25:
53 Meltdowns can be permanent or transitory as we have seen these may result from sample contamination effectsDoes the capillary need to be replaced?No! The next injection looks fine…
54 Effect of contaminant in reference sample Contamination results in problems in subsequent analysesSnow stormEffect is transitory
55 Effect of Ni Cations on a DNA Separation Ni-counterion TH01 (pH 7)Ni-intercalated TH01 (pH 8.3)1 l TH01 added to 10 l of 3.0 mM NiCl2 in 10 mM Tris, pH 7 or pH Sample allowed to interact for 1 hr and then 1 l added to ROX/formamide.
56 Transition metal ionsMetal cations present in degraded samples represent a different type of contaminationZn2+, Co2+, and Ni2+ form DNA-metal ion complexes, termed M-DNA.at pH conditions above 8,These cations produce severe effects in CE injection and analysisHartzell and McCord, Electrophoresis, in pressA proposed schematic for Zn-DNA11) Rakitin, A. et al. Phys. Rev. Lett. 2001, 86,
57 CE: Effect of pH 7 vs. 8.3Ni-counterion TH01 (pH 7)Ni-intercalated TH01 (pH 8.3)1 l TH01 added to 10 l of 3.0 mM NiCl2 in 10 mM Tris, pH 7 or pH Sample allowed to interact for 1 hr and then 1 l added to ROX/formamide.
58 Meltdowns may be the result of Bad formamideExcess salt in sample/renaturationWater in the polymer bufferSyringe leak or bottom outPoisoned capillaryConductive polymer buffer due to urea degradationCrack/shift in capillary windowDetergents and metal ions
59 A permanent loss of resolution may mean Adsorptive sites on a capillaryInitiation of electroosmotic flowConductivity changes in bufferWrong molecular weight or concentration of sieving polymer (viscosity)
60 5. Troubleshooting benchmarks Monitor run currentObserve syringe position and movement during a batchExamine ILS (ROX) peak height with no sampleObserve “250 bp” peak in GS500 size standardMonitor resolution of TH01 9.3/10 in allelic ladder and size standard peak shapesKeep an eye on the baseline signal/noiseMeasure formamide conductivityReagent blank – are any dye blobs present?See if positive control DNA is producing typical peak heights (along with the correct genotype)
61 Measurement of Current V/I = R where R is a function of capillary diameter, [buffer], and buffer viscosityIn a CE system the voltage is fixed, thus changes in resistance in the capillary will be reflected in the current observedAir bubbles, syringe leaks, alternate paths to ground, changes in temperature, changes in zeta potential, and contamination, will be reflected in the currentA typical current for a CE system with POP4 buffer is 8-12 µA (microamps)
62 Syringe TravelThe ABI 310 instrument also keeps track of the position of the syringe (in the log file)Depending on the resistance to flow, the syringe will travel different lengthsSyringe leaks may be reflected in a longer distance traveled prior to each injectionThese leaks occur around the barrel of the syringe and at the connection to the capillary block
63 Use of ABI 310 Log File to Monitor Current and Syringe Travel Syringe PositionCurrent
64 Measurement of Resolution for QC % Valley (works best if peak heights are equal)%valley = x 1 – (Ave peak height/height to valley)For the THO1 sample value = 0%For the sample below, value = 20%9.310Chromatographic ResolutionThe proper way to measure thisExpand the scale and focus on two peaksR = 1.18 (W1/2 + W1/2)/DResolution in basepairs =Distance between peaks/Rie for R=2 and distance between peaksIs 4 then resolution is 2 base pairsTHO /10DW1/2WROX Ladder
65 ROX Ladder QC procedures A recommended sequence for initial operation of the 310Rox ladder – initial injection - throwawayRox ladder- QC to test peak intensity and look for problems in blankAllelic ladder- to determine resolution and to provide standard10-15 samplesAllelic ladder
66 Voltage Spikes in Rox Ladder demonstrates the value of running the ladder by itselfBlank ladder with current spikes. Note enhanced intensity
67 Measurement of Signal and Noise Ratio You can also use the ROX size standard to keep track of sensitivityFor a given set of runs determine the average peak height of the Rox standardMonitoring this signal level will help determine if any major loss of sensitivity has occurredYou can also measure the P-P noise level in the same way and compare the two values.
69 Question: What is a real blank? Because of the stacking effect, injections of pure water or formamide can produce extreme sensitivityThis will allow you to detect small amounts of DNA clinging to the capillary, leading to a false impression that carry-over is a problemInstead, inject ROX plus formamide as your blank. In this case the added salt and fluorescent DNA drowns out these spurious peaks
70 Measuring Formamide Conductivity (not this way)The key is to measure the bottle when it comes in or buy the good stuff and immediately pipette it out into small tubes with or without ROX already added. Then freeze the tubes.Do not ever open a cold bottle of formamide. Water will condense inside and aid in the formation of conductive formic acid.
71 Conclusion: Troubleshooting is more than following the protocols It means keeping watch on all aspects of the operation1. Monitoring conductivity of sample and formamide2. Keeping track of current and syringe position in log.3. Watching the laser current4. Watching and listening for voltage spikes5. Monitoring room temperature and humidity