One-Tube MLPA Protocol for DNA Detection and - MRC
Contents
1. d Impurities in the test or reference DNA samples including NaCl or KCI gt 40 mM and other salts phenol IA moO ora ee ethanol heparin EDTA gt 1 5 mM and Fe The concentration of impurities will increase due to evaporation of sample DNA or by SpeedVac concentration of sample DNA do not use this Use of low quality plastics as these may leak impurities such as releasing agents into the MLPA reaction Depurination of sample DNA during the initial 98 C heat treatment This can occur when the sample has insufficient buffering capacity sample dissolved in dH O instead of TE A minimum of 5 mM Tris pH 8 2 in the sample DNA solution is required Sample DNA denaturation problems causing part of the DNA template to be unavailable for the MLPA probes Certain DNA purification methods e g Qiagen EZ1 result in a high salt concentration in DNA samples Extremely GC rich regions are not denatured at 98 C when more than 40 mM NaCl or KCI is present Use of whole genome amplification WGA of sample DNA due to its amplification bias Use of incorrect DNA quantities Use of insufficient or unsuitable reference samples Improper mixing of enzyme solutions e g by mixing insufficiently or too vigorously Deviating probe ratios that are due to harmless SNPs mutations and small indels within the sequence detected by a probe Evaporation during o n hybridisation causing increased salt concentration and strong sample DNA secondary str2. 2 ul PCR Primer mix 0 5 ul SALSA Polymerase Troubleshooting a detailed Quality Control Flowchart is available on www mlpa com PREVENT FALSE POSITIVE OR NEGATIVE RESULTS READ THE COMPLETE PROTOCOL AND STRICTLY FOLLOW THIS PROTOCOL READ THE MOST RECENT VERSION OF THE PRODUCT DESCRIPTION www mlipa com page 12 of 12
3. DNA denaturation can result in false positive deletions NOTE When using ABI POP7 polymer a non specific fragment of 87 nt is usually present which may coincide with the 88 nt D fragment gt D FRAGMENTS LOW SAMPLE DNA DENATURATION INCOMPLETE DNA in TE P335 probemix DNA in TE 40 mM salt P335 probemix DNA in TE 100 mM salt P335 probemi 160 12 Size Size Size Figure 4 Effect of poor denaturation on D fragments D fragments are low when sample DNA denaturation is incomplete here induced by adding salt MLPA results on DNA sample containing in A TE B TE 40 mM NaCl C TE 100 mM NaCl 7 2 NO DNA CONTROL In a typical No DNA control only the four Q fragments are visible However MLPA PCR reactions are more prone to non specific peaks in the No DNA Control than a normal PCR In some probe mixes a few peaks may be visible in No DNA Controls These non specific peaks should not influence MLPA results when sufficient sample DNA is used they should be outcompeted by the MLPA probes similarly to the Q fragments in figure 3 Notify MRC Holland in case a non specific peak in the No DNA control is reproducibly higher than the Q fragments 7 3 EVAPORATION PROBLEMS Evaporation may occur during A pipetting the ligation reaction at 54 C or B overnight hybridization In case you Suspect evaporation problems the following may help A Reduce handling time by using multi channel pipettes B Test evaporation by in
4. is added to the sample Each MLPA probe consists of two oligonucleotides that must hybridise to immediately adjacent target sequences in order to be ligated into a single probe Each probe in an MLPA probemix has a unique amplicon length typically ranging between 130 500 nt During the subsequent PCR reaction all ligated probes are amplified simultaneously using the same PCR primer pair One PCR primer is fluorescently labelled enabling the amplification products to be visualised during fragment separation This is done on a capillary electrophoresis instrument yielding a specific electropherogram Figure 2 left The relative height of each individual probe peak as compared to the relative probe peak height in various reference DNA samples reflects the relative copy number of the corresponding target sequence in the sample A deletion of one or more target sequence thus becomes apparent as a relative decrease in peak height Figure 2 right while an increase in relative peak height reflects an amplification SAMPLE DNA PROBE PROBE AMPLIFICATION OF DENATURATION HYBRIDISATION LIGATION LIGATED PROBES Figure 1 MLPA reaction Electropherogram reference sample A A P oP K z gt ah P ee rt G z E r a ni gt f A srg y s TYG y 4 yea eSeoas amp ges z Q lt i NEJuRyug 3 Ke ss g 15 e T es o Sw n gt lt A 7 u Swltws SY gt v gt p f 7 FSF OES 3 o OTE T o e2rekin
5. min at 98 C cool samples to 25 C before removing tubes from thermocycler 5 4 HYBRIDISATION REACTION DAY 1 Vortex MLPA buffer and MLPA probemix before use Prepare hybridisation master mix containing for each reaction 1 5 ul MLPA buffer yellow cap 1 5 ul probemix black cap Mix hybridisation master mix well by pipetting or vortexing After DNA denaturation add 3 ul hybridisation master mix to each sample tube Mix well by pipetting up and down Continue thermocycler program incubate for 1 min at 95 C then 16 20 hours at 60 C 5 5 LIGATION REACTION DAY 2 Vortex the two Ligase Buffers before use Prepare a Ligase 65 master mix For each reaction mix 25 ul dH O 3 ul Ligase Buffer A transparent cap 3 ul Ligase Buffer B white cap Then add 1 ul Ligase 65 enzyme green cap Mix well by pipetting gently up and down Never vortex enzyme solutions Continue thermocycler program pause at 54 C When the samples are at 54 C add 32 ul ligase master mix to each tube Mix by gently pipetting up and down Add the ligase master mix while the samples are IN the thermocycler Continue thermocycler program 15 min incubation at 54 C for ligation 5 min at 98 C for heat inactivation of Ligase 65 enzyme Pause at 20 C At this point tubes can be removed from the thermocycler 5 6 PCR REACTION DAY 2 Vortex SALSA PCR primer mix Warm polymerase for 10 sec in your hand to reduce viscosity Prepare polymerase mas
6. or capillaries POSITIVE CONTROL SAMPLES Including positive controls is recommended when possible When using cell line DNA note that this may have acquired additional copy numbers incl complete chromosomal gains losses ALIQUOT PRECIOUS REFERENCE CONTROL SAMPLES and store these at 20 C Contamination with microorganisms or moulds can deteriorate samples that are stored at 4 C for an extended period MLPA REACTION DNA DETECTION QUANTIFICATION NOTES TO READ BEFORE YOU START DAY 1 Use a calibrated thermocycler with heated lid 99 105 C Always vortex thawed buffers and probemix before use MLPA buffer is typically frozen at 20 C but may remain liquid due to its high salt concentration Centrifuge all MLPA reagent tubes for a few seconds before use as drops may have adhered to the lid Enzyme solutions contain 50 glycerol and remain fluid at the recommended storage temperature Never vortex enzyme solutions Master mixes containing enzymes should be mixed by gently pipetting up and down When the viscous enzyme solution is not mixed properly with the buffers unreliable results will occur If the enzyme solution is mixed too vigorously however enzyme inactivation occurs When preparing master mixes always add enzymes last To minimise sample variation prepare sufficiently large volumes of master mix solutions Include a 5 10 volume surplus to allow for pipetting errors Prepare Ligase 65 master mix and Polymerase master mix lt
7. 1 hour before use and store on ice When running a large number of samples use multi channel pipettes to avoid excessive evaporation In this General MLPA protocol the complete 40 ul ligation reaction is used for the PCR The alternative two tube MLPA protocol which uses only 10 ul of the ligation reaction for the PCR is available on www mlpa com The two tube protocol may have advantages when using DNA samples containing impurities such as high EDTA concentrations gt 1 5 mM or PCR inhibitors A vial of PCR buffer required for this alternative protocol can be ordered for free The two tube protocol is for research use only RUO not for in vitro diagnostic use IVD www mlpa com page 6 of 12 MRC Holland_ See MLPA MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 5 2 THERMOCYCLER PROGRAM FOR THE MLPA REACTION 1 DNA denaturation 1 98 C 5 minutes 2 259C pause 2 Hybridisation reaction 3 95 C 1 minute 4 60 C pause 3 Ligation reaction 54 C Pause 54 C 15 minutes 98 C 5 minutes 8 20 C pause 4 PCR reaction 9 35 cycles e 95 C 30 seconds e 60 C 30 seconds e 72 C 60 seconds 10 72 C 20 minutes 11 15 C pause 5 3 DNA DENATURATION DAY 1 Label 0 2 ml tubes strips or plates Add 5 ul DNA sample 50 250 ng 50 100 ng is optimal to each tube Use TE for no DNA control Place tubes in thermocycler start MLPA thermocycler program see above Denature sample DNA for 5
8. DOSAGE QUOTIENT DQ The absolute fluorescence measured by capillary electrophoresis cannot be used directly for copy number calculations as it is affected by many variables First each probe s measured fluorescence must be normalised within each sample to get meaningful data Second various samples need to be compared to establish which sample has aberrant copy number changes Therefore MLPA normalisation consist of 2 steps intrasample normalisation comparison of probe peaks WITHIN the sample and intersample normalisation comparison or relative probe peaks BETWEEN samples 1 Intrasample normalisation Within each sample compare each probe peak TO the peaks of the reference probes Reference probes detect sequences that are expected to have a normal copy number in all samples Almost all MLPA probemixes contain 8 or more reference probes located on various chromosomes The relative probe signals determined in step 1 are then used in 2 Intersample normalisation Final probe ratios are determined by comparing the relative probe peak in the DNA sample of interest TO all reference samples Reference DNA samples are expected to have a normal copy number for both the reference and target probes The MLPA peak pattern of a DNA sample without genomic abnormalities will be identical to that of reference samples final probe ratios determined in step 2 will be 1 0 For heterozygous deletions probe ratios will be 0 5 This final probe ratio is a
9. I3500 50 9 ul HiDi formamide injection time 15 sec ABI 3500 xL cm Seal the injection plate heat 3 min at 86 C run voltage 15 kV ABI 3730 xL cool for 2 min at 4 C F run time 1800 s oven temperature 60 C polymer POP4 or POP7 ABI Prism 310 0 75 ul PCR reaction injection voltage 1 6 kV 0 75 ul dH2O injection time 15 sec 0 5 ul GS 500 ROX LIZ size standard filter set D 13 5 ul HiDi formamide polymer POP4 Heat 3 min at 86 C cool for 2 min at 4 C ABI 3130 3500 amp 3730 have been validated and found suitable to be used with IVD registered MLPA products Briefly heating the injection mixture before capillary electrophoresis is essential 7 MLPA QUALITY CONTROL AND TROUBLESHOOTING For analysis of MLPA data use Coffalyser Net software which can be freely downloaded from www mlpa com The Coffalyser Net software provides quality scores for each reaction and helps with interpretation of the control fragments that are included in each SALSA MLPA probemix 7 1 MLPA QUALITY CONTROL FRAGMENTS Almost all SALSA MLPA probemixes contain nine control fragments as described below Internal quality control fragments Length nt Interpretation 92 nt benchmark 92 Normal probe forms a benchmark to compare other quality control probe fragments to High when DNA amount is too low or ligation failed All four Q fragment signals gt 33 of the 92 nt control fragment gt DNA quantity insufficient Low in case of poor sample D
10. MRC Holland MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 MRC Holland MLPA General Protocol Instructions For Use General MLPA protocol for the detection and quantification of nucleic acid sequences To be used in combination with the appropriate MLPA probemix specific product description Certain MLPA products from MRC Holland are registered for In Vitro Diagnostic IVD use but only in specific countries In all other cases MLPA products are for Research Use Only RUO Information on IVD registration can be found in the appropriate probemix specific product description An alternative two tube protocol is available which is only recommended for use on low quality DNA samples RUO Separate protocols exist for the detection of DNA methylation status MS MLPA and RNA expression RT MLPA These protocols are available on www mlpa com onal Manufacturer MRC Holland B V Willem Schoutenstraat 1 1057 DL Amsterdam the Netherlands Website www mlpa com Phone 31 888 657 200 E mail info mlipa com information amp technical questions order mlpa com for orders coffalyser mlpa com for Coffalyser related questions www mlpa com page 1 of 12 MRC Holland hree a MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 MLPA General Protocol Document History Version MDP 005 22 SEPT 2014 In section 3 3 Storage and Stability the sentence Ligase buffer A should not be exposed to mor
11. NA denaturation Signal lt than 40 of the 92 nt control fragment gt DNA denaturation problems X amp Y fragments 100 105 Control for sample swapping Q fragments 64 70 76 82 D fragments 88 96 t Rare cases are known of males lacking this Y specific sequence and females carrying this Y sequence on an X chromosome www mlpa com page 8 of 12 MRC Holland nner a MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 QUANTITY CONTROL FRAGMENTS Q FRAGMENTS The four Quantity Fragments Q fragments at 64 70 76 82 nt are complete fragments that do not need to hybridise to DNA or be ligated to be amplified during PCR The more sample DNA is added the lower they get see Figure 3 gt ALL FOUR Q FRAGMENTS HIGH INSUFFICIENT SAMPLE DNA USED OR LIGATION REACTION FAILED 64707682 Figure 3 Effect of DNA quantity on Q fragments The more sample DNA is used the lower the Q fragments MLPA results with A 5 ng DNA B 10 ng C 50 ng DENATURATION CONTROL FRAGMENTS D FRAGMENTS The two DNA Denaturation Fragments D fragments 88 amp 96 nt detect sequences in exceptionally strong CpG islands CpG islands have a high GC content and are difficult to denature When the 88 and 96 nt fragments are low lt than 40 of the 92 nt control fragment this indicates denaturation problems of the sample DNA A poor denaturation can be due to presence of gt 40 mM salt in the DNA sample Incomplete sample
12. OMPONENTS PROVIDED PER SALSA MLPA REAGENT KIT i Volumes AEn lhe Ingredients component EK1 EK5 SALSA MLPA Buffer 1 yellow cap 180 ul 5x180 ul KCI Tris HCI EDTA PEG 6000 oligonucleotide SALSA Ligase 65 Glycerol EDTA Beta Mercaptoethanol KCI Tris HCI nonionic detergent Ligase 65 2 115 ul 5x115 ul bee green cap enzyme bacterial origin Ligase Buffer A a 3 transparent cap 360 ul 5x360 ul Coenzyme NAD bacterial origin Ligase Buffer B ie eae 4 white cap 360 ul 5x360 ul Tris HCl MgCl2 non ionic detergent 5 SALSA PCR Primer 240 ul 5x240 ul Synthetic oligonucleotides with fluorescent dye FAM or Cy5 dNTPs Tris HCI KCI Mix brown cap H H EDTA nonionic detergent 6 SALSA Polymerase 65 ul 5x65 ul Glycerol non ionic detergents EDTA DTT KCI Tris HCI Polymerase enzyme orange cap H H bacterial origin 7 MLPA General protocol None of these ingredients are derived from humans animals or pathogenic bacteria Based on the concentrations present none of the ingredients are hazardous as defined in the Hazard Communication Standard An MSDS is not required for these products none of the preparations contain dangerous substances as per directive 67 548 EEC amp amendments at concentrations requiring distribution of MSDS as per directives 1999 45 EEC 2001 58 EEC 3 3 STORAGE AND STABILITY All components of the SALSA MLPA reagent kit must be stored directly upon
13. PA DATA NORMALISATION I FROM PEAKS to DOSAGE QUOTIENT DQ 10 8 3 PRINCIPLES OF MLPA DATA NORMALISATION II FROM DOSAGE QUOTIENT TO COPY NUMBER 10 9 INTERPRETATION OF RESUEN S sxccevavesses cenccnnceseGarecsecueyecendeteveanvenwsenveyerietuansetonshaneneta lias yescuaveesneniaaeestanes 11 10 MEPA PROTOCOL IN A NUT SHELL sesciccncccawsisateanectevpessansesadensaeswousavanrnenautsienkveauseeanthnuesiaeusaenreasecsieesienvanes 12 www mlipa com page 2 of 12 MRC Holland mo MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 1 INTENDED USE Copy Number Variations CNVs are a prominent source of genetic variation in human DNA and play a role in a wide range of disorders Multiplex Ligation dependent Probe Amplification MLPA is a semi quantitative technique that is used to determine the relative copy number of up to 60 DNA sequences in a single multiplex PCR based reaction MRC Holland manufactures and sells MLPA reagents and a wide range of MLPA probemixes Together these can be used to detect deletions and duplications in a DNA sample Details on the intended use are specified in the MLPA probemix specific Product Description 2 MLPA ASSAY PRINCIPLE As outlined in Figure 1 the principle of MLPA is based on the amplification by use of a single PCR primer pair of up to 60 probes each of which detecting a specific DNA sequence of approximately 60 nt in length After denaturation of the sample DNA a mixture of MLPA probes
14. TS PROVIDED PER SALSA MLPA REAGENT KIT cscccsseccccsecccseccscececseeeaseeeansess 4 3 3 STORAGE AND STABILITY cccccccseccccscccccccccseecesecuucsecueueecaueuusueuecusuuaseueaueuauueueaeuunasess 4 3 4 MATERIALS REQUIRED BUT NOT PROVIDED cccceceeceeeceeeeceeeceeeeceeeaueeeaaaeeesneeteneeteeerers 4 3 5 PRECAUTIONS AND WARNINGS cccccccccccscccccseccuseccucsecuecuecauceceseueeusuecseuuuausuuaueueeuenaneees 4 3 6 LIMITATIONS OF THE PROCEDURE cccccsccccssecccceceucsccucccecaececeuseeeusecuseuuuueuaaeueuaesenaeess 5 4 ASSAY SETUP INSTRUCTIONS cccccesecccseccesececscececceuaucuceseuecsecuuseuuuauaususueususuusesuceusueuadsueaauauaaeuensuunneeees 5 4 1 SAMPLE TREATMENT cccccsssecccceccccsccccceccuscecusseecuseecussuuaseuueseuuaueeusueueeusunauseuuansueaeeenens 5 4 2 SELECTING REFERENCE amp OTHER CONTROL SAMPLES csscccecccccscececceccuseceseeuaeseeaeeeeeas 6 5 MLPA REACTION DNA DETECTION QUANTIFICATION cccccccceceeseeeeeeeeeceeeeeceeceeeeeeeeceeeeauasauaasaaaaeeeseeesess 6 5 1 NOTES TO READ BEFORE YOU START DAY 1 cccccccccecceceeeceeeeeeeeeeeeeeeseeeeeeeeeeeeeeeseeeeeeeeeeeens 6 5 2 THERMOCYCLER PROGRAM FOR THE MLPA REACTION cccccsscccccsecccseecssccuececeaueeuaseenensess 7 5 3 DNA DENATURATION DAY 1 vccousevsscuciccesaiveasdaviorturnievunasesseveverinesevnsiaivvoowactvselayseeedseeues 7 5 4 HYBRIDISATION REACTION DAY 1 ccccccccccecceeeceeceeeececeeeeeseeeeeeeeseeeeeeeseeeeeeeeeteeese
15. arrival between 25 C and 15 C Shielded from light and in the original packaging When stored under the recommended conditions a shelf life of at least 1 year is guaranteed also after opening See the labels on each vial for the exact expiry date of each reagent Reagents should not be exposed to more than 25 freeze thaw cycles 3 4 MATERIALS REQUIRED BUT NOT PROVIDED SALSA MLPA Probemix and corresponding Product Description Ultrapure water TEO 1 10 mM Tris HCl pH 8 2 0 1 mM EDTA Thermocycler with heated lid 99 105 C and standard laboratory equipment PCR tubes strips or plates High quality formamide e g Hi Di Formamide Applied Biosystems 4311320 Capillary Electrophoresis equipment Beckman Coulter or Applied Biosystems Labelled size standard o Beckman CEQ DNA Size Standard Kit 600 p n 608095 o Applied Biosystems GeneScan 500 LIZ 4322682 or 500 ROX 4310361 e Polymers o Beckman GenomeLab Linear Polyacrylamide LPA denaturing gel p n 391438 o Applied Biosystems preferred POP 4 4352755 or POP 7 4352759 POP 6 is not recommended because of its high resolution though for some older sequencers POP 6 may be the only option e Coffalyser Net analysis software and User manual and product specific data analysis sheet 3 5 PRECAUTIONS AND WARNINGS e For in vitro use only e Always consult the most recent version of the relevant probemix specific product description AND this MLPA General protocol Stric
16. cubating 8 ul H2O overnight at 60 C gt 5 ul H2O should remain To reduce evaporation 1 ensure heated lid works well 2 increase decrease pressure of lid on tubes 3 try different tubes e g Thermo Fischer ABgene AB 0773 AB 0451 4 use mineral oil Vapor lock Qiagen 981611 add small drop of oil to DNA sample just enough to cover it There is no need to remove oil After addition of MLPA buffer probemix mixture or polymerase mix centrifuge very briefly After addition of ligase mix gently pipet up and down 7 4 TROUBLESHOOTING A detailed Quality Control Flowchart is available on www mlipa com and can be used for quality control and troubleshooting www mlpa com page 9 of 12 MRC Holland mo MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 8 PRINCIPLE OF MLPA DATA ANALYSIS 8 1 COFFALYSER NET FOR MLPA DATA ANALYSIS Use Coffalyser Net software for MLPA data analysis The Coffalyser Net Manual provides a step by step instruction on MLPA data analysis Both software and manual are freely downloadable on www mlipa com This section describes the basic principles of MLPA analysis Coffalyser Net analysis is based on these principles but uses a more robust algorithm In addition Coffalyser Net selects the best analysis method for each MLPA probemix and offers extensive quality control For IVD registered MLPA applications Coffalyser net must be used 8 2 PRINCIPLES OF MLPA DATA NORMALISATION I FROM PEAKS to
17. e PCR protocol which uses only part of the ligation reaction for the PCR see section 5 1 MLPA is more sensitive to contaminants than simple monoplex PCR assays Contaminants left after DNA extraction listed in 3 6 may influence MLPA performance To minimise their effect ensure the extraction method tissue type DNA concentration and treatment are as identical as possible in test and reference samples EDTA concentration of the samples should not be higher than 1 5 mM Sample DNA should not be concentrated by evaporation or SpeedVac as this leads to a very high EDTA concentration Heparinised blood is not preferred for DNA extraction Heparin is difficult to remove from DNA preparations and can distort the MLPA PCR reaction Certain DNA purification methods e g Nucleospin gDNA Clean up XS are capable of removing heparin contamination Do not use Qiagen M6 M48 and M96 systems for DNA purification high salt concentrations For Qiagen EZ1 only use the QIAGEN Supplementary Protocol for use in Third Wave Invader assays see www mlpa com DNA from whole genome amplification reactions WGA is not suitable for MLPA because of its amplification bias MRC Holland has tested and can recommend the following extraction methods Qiagen Autopure LS automated and QlAamp DNA mini midi maxi kit manual Promega DNA extraction Wizard manual Salting out manual www mlpa com page 5 of 12 MRC Holland_ See MLPA MLPA DNA Pr
18. e than 20 freeze thaw cycles has been replaced by Reagents should not be exposed to more than 25 freeze thaw cycles The word IVD certified has been replaced by IVD registered p 10 Version MDP 004 11 JUL 2014 Information on IVD registration added 3 5 Precautions amp Warnings and 3 6 Limitations of the Procedure added Information presented more concisely Quality Control Flowchart removed 8 Data Analysis section rewritten Version MDP 003 08 AUG 2013 Document COMPLETELY rewritten New Quality Control Flowchart added Information presented more concisely Version MDP 002 23 JAN 2012 Sentence removed that the new PCR primer mix has a lot no of F44 or newer Old PCR primer mix cannot be recognised by the lot no but by the absence of the MRC Holland logo on its label Version MDP 001 17 JUN 2011 NEW DOCUMENT Previous MLPA General Protocol completely rewritten for use with the one tube protocol Table of Contents 1 INTENDED U E orr E E csi deeseaedersantuetessseneaneescsecdsdateecseceieraeessaiitupanneres eames 3 2 MLPA ASSAY PRINCIPLE ccssseccececcccsccccccccusceceseuceseucussuuuseuueusuuauuausususeuseneseusuausuuaeueussuueuseuueaseusnsenaaess 3 3 SALSA MLPA ASSAY COMPONENTS amp STORAGE CONDITIONG cccccccssccecseeccceccccsccacceccucueueseuceususasseeensss 3 3 1 REAGENT KIT ITEM NUMBERS cccccccccccsecccccecceseccucsecuaceecauceususueeusutaessuuaueuaeueuaeseananeess 3 3 2 COMPONEN
19. effect on MLPA results Change capillaries and polymer regularly Polymer quickly deteriorates after prolonged exposure to gt 25 C In case size standard peaks are low and broad the capillaries or polymer are usually deteriorated Formamide can become acidic Use high quality formamide and store it in aliquots at 20 C In case all MLPA peaks are low do not add more MLPA PCR product to injection mixture Adding more PCR product increases the salt concentration in the injection mixture which competes with DNA for injection When an increase in peak heights is desired increase injection time voltage None of the peaks should be off scale Reset the bin settings in the fragment analysis software whenever using a different a MLPA probemix lot b size standard c capillary electrophoresis apparatus or d run settings 6 2 ELECTROPHORESIS SPECIFICATIONS Instrument Primer Capillaries Injection mixture Initial Settings Beckman Cy5 33 cm 0 7 ul PCR reaction run method Frag CEQ 2000 0 2 ul CEQ size standard 600 capillary temperature 50 C CEQ 8000 32 ul HiDi formamide Beckman SLS denaturation 90 C for 120 sec CEQ 8800 Add one drop of high quality mineral oil injection voltage 1 6 kV GeXP injection time 30 sec run time 60 min run voltage 4 8 kV FAM ABI Prism 3100 36 50 cm 0 7 ul PCR reaction run module FragmentAnalysis Avant 0 3 ul ROX or 0 2 ul LIZ GS 500 size standard injection voltage 1 6 kV ABI 3130 xL AB
20. fluence the probe signal are diluted An MLPA result is unlikely to be reliable when e Probes for non neighbouring exons show a decreased or increased signal e g deletion of exon 3 and 17 e Inthe same sample one or more reference probes show an abnormal copy number e Copy number changes are detected with an unusually high frequency in a patient cohort for a certain disease 10 MLPA PROTOCOL IN A NUTSHELL SAMPLE DNA PROBE PROBE AMPLIFICATION OF DENATURATION HYBRIDISATION LIGATION LIGATED PROBES 1 DNA DENATURATION e Heat a5 ul DNA sample for 5 minutes at 98 C 2 HYBRIDISATION OF PROBES TO SAMPLE DNA e Cool down to room temperature open the tubes e Add a mixture of 1 5 pl SALSA probemix and 1 5 ul MLPA buffer and mix e Incubate 1 minute at 95 C 16 hours hybridisation at 60 C 3 LIGATION OF HYBRIDISED PROBES e Lower thermocycler temperature to 54 C open tubes e Add 32 ul ligase master mix incubate 15 minutes at 54 C e Heat to inactivate the ligase enzyme 5 minutes 98 C 4 PCR AMPLIFICATION OF LIGATED PROBES e Cool down to room temperature open tubes e Add 10 ul polymerase master mix at room temperature e Start PCR 5 CAPILLARY ELECTROPHORESIS OF PCR PRODUCTS 6 ANALYSE RESULTS e Determine RELATIVE size of the fluorescent peaks within each sample e Compare these results to reference samples Ligase master mix 3 ul Ligase Buffer A 3 ul Ligase Buffer B 25 ul water 1 ul Ligase 65 Polymerase master mix 7 5 ul water
21. lso called Dosage Quotient DQ Coffalyser Net calculates the DQ for each probe in each sample Probes should be arranged based on chromosomal location for correct interpretation this will also aid in detecting subtle changes such as mosaicism 8 3 PRINCIPLES OF MLPA DATA NORMALISATION ITI FROM DOSAGE QUOTIENT TO COPY NUMBER How to discriminate normal deletion and duplication results Table 1 gives the relationship between copy number status and the typical distribution of DQs based on a large number of samples at MRC Holland Table 1 Relation Dosage Quotients and Copy Number based on normal status of 2 copies Dosage Quotient Distribution Copy Number Status 0 copies homozygous deletion QO 0 40 lt DQ lt 0 65 2 gt 1 copy heterozygous deletion 0 80 lt DQ lt 1 20 NORMAL identical to reference samples 1 30 lt DQ lt 1 65 2 gt 3 copies heterozygous duplication 1 75 lt DQ lt 2 15 2 gt 4 copies or 1 gt 2 copies All other values Ambiguous result In addition the standard deviation of each probe should be measured to ensure the result is reliable Coffalyser Net software considers a result as statistically significant change if Furthermore to be indicative of a heterozygous deletion duplication respectively the following applies 2 DQzrobe 1 sample a Below O 7 or above 1 3 indicates a heterozygous deletion duplication respectively k Coffalyser Net starts with raw data analysis baseli
22. n 3 copies of chromosome 21 70 contain 2 copies The DQ found for chr 21 probes will be 30 x 3 70 x 2 2 1 15 2 number of copies present in reference samples 6 When designing probes known SNPs are avoided when possible However new SNPs are continuously being discovered Please notify us when a polymorphism or a frequent pathogenic mutation influences a probe signal 7 Schwartz M and Dung M 2004 Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation dependent probe amplification method Genet Test 8 361 7 www mlpa com page 11 of 12 MRC Holland me MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 e Most MLPA probemixes contain reference probes Copy number changes detected by reference probes are unlikely to be related to the condition tested for The identity of reference probes is available on request e Incertain cases analysis of parental samples might be necessary for correct interpretation of results e Internal MLPA validation in your laboratory is essential MLPA results are more reliable when e The overall standard deviation of each probe in the reference samples is low lt 10 e Probes show a decreased or increased signal for adjacent exons multi exon deletion or duplication e The same result is obtained in a new MLPA run using less DNA if possible or using different reference samples When less DNA is used any possible contaminants which may in
23. n with its statistical significance in the experiment such a partial copy number change can still be recognised if a the experiment was performed well and b a similar DQ was obtained for adjacent probes 9 INTERPRETATION OF RESULTS The information provided in the MLPA probemix specific Product Description is essential for a correct interpretation of MLPA results To judge whether obtained results are reliable and to interpret MLPA results correctly a good understanding of the MLPA technique and the application tested for is essential Keep the following in mind e For most applications the major cause of genetic defects will be small point mutations most of which will not be detected by MLPA MLPA will not detect most inversions translocations nor copy number changes that lie outside the sequence detected by the MLPA probes MLPA probemixes cannot detect deletions duplications lying outside the target sequence of its probes MLPA probes typically detect a sequence of 55 80 nt Most product descriptions mention only partial probe sequences Complete probe sequences are available on www mlpa com e Sequence changes SNPs point mutations in the probe s target DNA sequence even when gt 10 nt from the ligation site can cause false deletions they may lower a probe signal by hampering ligation or by destabilising the binding of the probe oligos to the sample DNA e Copy number changes detected by a single probe always require confirma
24. ne correction peak identification and extensive quality control e g DNA quantity used complete DNA denaturation degree of sloping Slope correction of the peak pattern is one of the many options 3 The standard deviation differs per probe and depends on many factors including 1 quality of DNA samples purity sample DNA fragmentation depurination other modifications 2 quantity of sample DNA used 3 number and quality of reference samples and positive samples 4 experimental conditions e g pipetting accuracy thermocycler temperature uniformity quality of amplicon separation and detection and 5 method used for data analysis Coffalyser Net evaluates ALL of these factors Coffalyser Net uses values of 0 7 and 1 3 as cut off values for a heterozygous deletion or duplication respectively when normal status is diploid Coffalyser Net thus uses sharp cut off values while table 1 gives a substantial ambiguous DQ range www mlpa com page 10 of 12 MRC Holland ee MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 For mosaic and tumour samples the 0 7 1 3 cut off lines of Coffalyser Net are not applicable as the DQs can take on any value their calculated DQs will depend on a the magnitude of the copy number change AND b the percentage of the different cell types in the DNA sample As Coffalyser Net determines the significance of a probe signal change by evaluating the magnitude of the calculated DQ n combinatio
25. nneeneeeenns 7 5 5 LIGATION REACTION DAY 2 ccccccccccccececeeceeeceeeeeeeeeeeeeeeeeeeeseeeeeeeseeeeeeeeeseeeeeeeeeneeeneeenneeens 7 56 PER RENCTION DAY essere ca reteecnancencecsadseecncencsesucancsenosustonstacadacdesteassessoqauuetssencsosscce 7 6 FRAGMENT SEPARATION BY CAPILLARY ELECTROPHORESIS csccccsscccescecscecucsecuseccucceuauuueuueueeauseeeuseuaeess 8 6 1 NOTES TO READ BEFORE YOU START csscccessecceseccececccececcuseecaceueeusuecseuuaseuaeueeuasunnaasess 8 6 2 ELECTROPHORESIS SPECIFICATIONS cscccesecccsecececcuececcasecessuceuseecsueueeseuaeuuueneueuaaess 8 7 MLPA QUALITY CONTROL AND TROUBLESHOOTING ccccccccccccecseeeeeeeeeeeceeeeeeeeceeeeeeeeceeesauassuaaneaeaeesseeesess 8 7 1 MLPA QUALITY CONTROL FRAGMENTS cccscsseeceeececeeeeeececeeceeeneeeeaaaaceaeesenecseeeeeeeeeeeseress 8 7 2 NO DNA CONTROL ccccccscecccecccccccccucecucsuscseecesecuusuuaauueuaueuesseueausueueseuaaseauaueneaeuenasens 9 7 3 EVAPORATION PROBLEMS ccccccsecccceccccscceceseecesscuacseuuauuecaueueaususeusecasetsueueuaaueunauunnaneees 9 7 4 TROUBLESHOOTING ccccececcsceccccccccucecuuceecuseecusscuuseuauueeuaueunseueausuuaseueuueuensuuusuunnaeess 9 8 PRINCIPLE OF MLPA DATA ANALYSIS c ccseccccsccccsececescececsccuesecuuseuuausueeusesauseuauesuuenuuuuaueuuauuunassueaessunaseeas 10 8 1 COFFALYSER NET FOR MLPA DATA ANALYSIS cccsssecececceccccaccecuuseeceseeaussecesseeasueaaeneas 10 8 2 PRINCIPLES OF ML
26. otocol version MDP 005 last revised on 22 SEPT 2014 4 2 SELECTING REFERENCE amp OTHER CONTROL SAMPLES 1 REFERENCE SAMPLES Reference samples should be included in each MLPA experiment Minor differences in experimental execution may affect the MLPA peak pattern Only compare samples that are a included in the same MLPA experiment b tested with the same probemix lot MULTIPLE REFERENCE SAMPLES are needed to estimate the reproducibility of each probe within each MLPA run Minimum is 3 different reference samples When testing gt 21 samples add 1 additional reference per 7 additional test samples Reference samples should be distributed randomly over the sample plate to minimise variation SELECTING REFERENCE SAMPLES Reference samples are DNA samples in which target and reference probe sequences are expected to have a normal copy number obtained from healthy individuals tissues They should be as similar as possible to test samples in all other aspects see 4 1 For formalin fixed paraffin embedded tissue use reference samples derived from similarly treated healthy tissue COMMERCIAL DNA In case of doubts about sample quality include one or more commercial DNA samples for comparison recommended Promega Cat Nr G1471 male G1521 female DNA NO DNA CONTROL Per MLPA run include a No DNA Control reaction replace 5 pl DNA by TE 10 mM Tris HCl pH 8 2 0 1 mM EDTA to check for contamination of TE MLPA reagents electrophoresis reagents
27. res Fi F i gt eee a O RREY oc Se a S es a fh amp i Seneca cs eum LEERE FLPE eo TT Ga UATATAUACLATAGLLBLTAATATATATATAWAWAVAUAUAWAVAUAUAUAWAVAULUAUAUAUAW ATA UIT j i SRT ORR PESP OE RSSRSSS ARE SELES SH ITEAL ETTET sete eee PUREST Soden Peds detspincsg Psa givetehe hhssener gods Electropherogram test sample 54s Figure 2 A Electropherogram of a test sample bottom is compared to that of a reference sample top showing a relative decrease of three probes in the test sample arrows B Calculated probe ratios of the same test sample as displayed by Coffalyser Net software after analysis of these two samples arranging probes by chromosomal location shows a reduced copy number for these three adjacent probes in the test sample LA gt 3 ey lil ashes bis oi i F F v 2 r na F Mapview Location SRR ALALILA LALALALA LLL ALLL L LALA L L iL Atul 3 SALSA MLPA ASSAY COMPONENTS amp STORAGE CONDITIONS 3 1 REAGENT KIT ITEM NUMBERS Description IEK1 FAM SALSA MLPA EK1 reagent kit 100 rxn FAM labelled PCR primer EK1 Cy5 SALSA MLPA EK1 reagent kit 100 rxn Cy5 labelled PCR primer IEK5 FAM SALSA MLPA EK5 reagent kit 500 rxn FAM labelled PCR primer EK5 Cy5 SALSA MLPA EK5 reagent kit 500 rxn Cy5 labelled PCR primer www mlpa com page 3 of 12 MRC Holland hme a MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 3 2 C
28. ter mix For each reaction mix 7 5 ul dH2O 2 ul SALSA PCR primer mix brown cap 0 5 pl SALSA Polymerase orange cap Mix well by pipetting up and down do not vortex Store on ice until use At room temperature add 10 ul polymerase master mix to each tube Mix by pipetting gently up and down Place the tubes in the thermocycler and continue the thermocycler program 35 cycles of 30 seconds 95 C 30 seconds 60 C 60 seconds 72 C End with 20 min incubation at 72 C pause at 15 C After the PCR reaction do not open tubes in the room with the thermocycler To avoid contamination use different micropipettes for performing MLPA reactions and handling MLPA PCR products PCR product can be stored at 4 C for 1 week For longer periods store between 25 C 15 C As fluorescent dyes are light sensitive store PCR products in dark box or wrapped in aluminium foil www mlpa com page 7 of 12 MRC Holland ee MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 6 FRAGMENT SEPARATION BY CAPILLARY ELECTROPHORESIS 6 1 NOTES TO READ BEFORE YOU START Size standard run conditions polymer fluorescent dye and volume of MLPA PCR reaction used depend on instrument type Settings below are standard settings Instrument settings may require optimisation for optimal MLPA fragment separation follow instructions of the manufacturer of the capillary electrophoresis apparatus Using old capillaries or polymer has a detrimental
29. tion Sequencing of probe target sequences may show that a lowered probe signal is caused by a mutation polymorphism The finding of two heterozygous sequences typically indicates that the sample DNA does contain two different alleles In contrast note that the finding of only a single rare allele by sequencing does not yet imply that the other normal allele is deleted the lower probe signal may be caused by a homozygous SNP even when the SNP is very rare e Long range PCR and qPCR are often used to confirm single exon deletions It is also possible to design your own synthetic MLPA probes for confirmation of results e g Hills A et al 2010 Mol Cytogenet 3 19 Information on MLPA probe design is available on www mlpa com for research use only e Not all deletions and duplications detected by MLPA are pathogenic MRC Holland cannot provide information whether a deletion or duplication of a specific exon will result in disease For many genes exons are described that are only present in certain transcript variants For some genes such as DMD in frame deletions resulting in mild or no disease have been described A duplication of one or more exons may disrupt that copy of the gene resulting in disease whereas a complete gene duplication may not be pathogenic Note that duplications that include the first or the last exon of a gene may leave one functionally intact copy e Germline copy number variations reported in healthy individ
30. tly follow this protocol e For professional use only Assay performance is dependent on operator proficiency and adherence to procedural directions Assay should be performed by professionals trained in molecular techniques e The person responsible for result interpretation should be aware of the latest scientific knowledge of the application in question and of known limitations of the MLPA procedure that may lead to incorrect results e Abnormalities detected by MLPA should be confirmed by an independent technique whenever possible especially if they concern a single MLPA probe e Internal validation of each MLPA application is essential include at least 16 normal DNA samples Validation should show a standard deviation lt 0 10 for each probe unless the relevant Product Description states otherwise Samples used for validation should be representative of samples used in daily practice www mlpa com page 4 of 12 MRC Holland_ See MLPA MLPA DNA Protocol version MDP 005 last revised on 22 SEPT 2014 3 6 LIMITATIONS OF THE PROCEDURE e For most MLPA applications the major cause of most genetic defects are small point mutations most of which will not be detected by MLPA probemixes MLPA will not detect most inversions balanced translocations nor copy number changes that lie outside or only partially inside the sequence detected by an MLPA probe e False positive or negative results can be caused by various factors including
31. uals can be found at http projects tcag ca variation Certain copy number aberrations can be due to somatic alterations For instance a somatic trisomy 12 is an early sign of Chronic Lymphocytic Leukemia CLL e In case of poor sample DNA denaturation even the apparent deletion of severa probes recognising adjacent genomic targets can be a false positive result The presence of salt in DNA samples e g gt 40 mM NaCl prevents DNA denaturation of GC rich regions Sequences in the vicinity of such CpG islands may denature at 98 C but will reanneal immediately upon cooling as the non denatured CpG island holds the two strands together Binding of probes to their target sequence in these regions will be hindered resulting in reduced signals for probes located within several kb from such strong CpG islands Always examine the D fragments carefully e MLPA tests provide the average copy number of the target sequences in the cells from which the DNA sample was extracted In case several probes targeting adjacent sequences have an unusual value but do not reach the usual threshold values for a deletion duplication mosaicism is a possible cause 0 65 lt DQ lt 0 80 1 20 lt DQ lt 1 30 However Coffalyser also calculates whether the DQ of a probe is statistically different from that in the reference population Coffalyser Net can therefore also be used on tumour samples and can identify some mosaic cases 5 Example In Sample A 30 of its cells contai
32. ucture This may prevent certain probes from binding to their target sequences Contamination during the MLPA reaction or electrophoresis with other PCR products Problems during capillary electrophoresis including saturation of the device causing peaks to be off scale Problems during normalisation including the use of incorrect normalisation algorithms or software An incorrect interpretation of results due to insufficient knowledge of the application or gene s in question Insufficient knowledge of the clinical effect of a found genetic aberration Not all deletions and duplications detected by MLPA may be pathogenic ASSAY SETUP INSTRUCTIONS 4 1 SAMPLE TREATMENT Use a total quantity of 50 250 ng preferably 50 100 ng of human DNA in a 5 ul volume for each MLPA reaction If necessary DNA samples can be concentrated by ethanol precipitation Glycogen Roche 901393 can be used as Carrier in ethanol precipitations More info on www mlpa com Dissolve and dilute sample DNA in TEo 1 10 mM Tris HCl pH 8 2 0 1 mM EDTA DNA preparations should contain 5 10 mM Tris buffer with a pH of 8 0 8 5 to prevent depurination during initial heat treatment at 98 C If unknown whether sufficient buffer is present add Tris HCl 4ul sample DNA iul 50mM Tris HCl pH 8 5 In case of doubts about DNA quality a use only 50 ng of sample DNA b clean contaminated samples by ethanol precipitation or silica based clean up kits c use the alternative two tub
Download Pdf Manuals
Related Search