Human and Clinical Genetics (LUMC) - LabJ_Protocols^©

MAPH - Multiplex Amplifiable Probe Hybridisation

(Stefan White/Margot Kalf; last modified October 4, 2002)

Abbreviations

Purpose

The following protocol is used to detect deletions and duplications in genomic DNA. The protocol has been modified from Armour et al. (2000) and has been published as White et al. (2002).

Protocol

This protocol assumes that you start with high molecular weight genomic DNA, at relatively high concentration (>0.5 ug/ul) and a probe mix with appropriate primers for amplification.

PREPARATION OF FILTERS WITH GENOMIC DNA

The test DNA is immobilized on small pieces of nylon filter (approx. 2 mm x 3 mm) after denaturation. Since many different DNA samples can be hybridised together at one time, you will need a way of distinguishing the different filters; one way to do this is to cut the corners of each filter in a distinctive pattern.

1. cut the filters from the nylon membrane, and arrange on a sheet of cling film on the bench. Mark the number of each filter next to it on the cling film
2. to 1 ug of each genomic DNA sample (plus controls), add 1 ul PCR-clean 1M NaOH. Spot this on the filter, 1 ul at the time, allowing the filter to dry between applications
3. when all the DNA has been applied to the filters, allow to dry and wrap up in the cling film
4. crosslink the DNA to the filters by UV irradiation (50 mJ, both sides, Stratalinker)

PRE-HYBRIDISATION AND HYBRIDISATION

1. prepare fresh pre-hybridisation solution
2. pre-hybridise the filters together in an Eppendorf tube containing 1 ml pre-hybridisation solution and incubate 2 h to O/N at 60^oC

HYBRIDISATION

1. prepare 300 ul pre-hybridisation solution, add 3 ul human Cot-1 DNA and boil 2 min in an Eppendorf tube
2. remove the pre-hybridisation solution from the filters, replace it with 200 ul of the boiled Cot-1/pre-hybridisation solution and incubate 30-60 min at 60^oC
3. prepare hybridisation solution;
   1. to 1 ul of probe-mix, add 1 ul Cot-1 DNA, 1 ul herring sperm DNA, 1 ul blocker-mix and 3 ul H₂O
      NOTE: details regarding availability of the probe set
   2. add 2 ul 1.0 M NaOH, incubate 1 min at 37^oC and place on ice
   3. add 3 ul 1.0 M NaH₂PO₄, mix and add the mix to the Eppendorf tube with the filters
      NOTE: up to 12 filters have been combined in one hybridisation successfully
4. incubate O/N at 60^oC

POST-HYBRIDISATION WASHES AND PCR

1. transfer the filters to a 50 ml tube containing 25 ml pre-warmed (60^oC) wash solution A
2. wash for a total of 45-60 min;
   1. 5 times at 60^oC in 25 ml pre-warmed (60^oC) wash solution A
   2. 5 times at 60^oC in 25 ml pre-warmed (60^oC) wash solution B
3. when washing is complete, tip out the filters into a Petri dish, identify each filter and place into its own PCR tube

FIRST PCR

The first stage in recovering the probes specifically bound to the filter involves a 5-cycle, 50 ul PCR in a thin walled tube. The filter is simply placed into the PCR-mix. The specially bound probes will be released during the first denaturation step and become available for subsequent amplification.

1. dispense 50 ul aliquots of PCR-mix A into thin-walled 200 ul PCR tubes and add the filter, transferring as little as possible of the washing solution
2. perform the first PCR; 5 min at 94^oC, 5 cycles of 45sec at 94^oC / 1 min at 57^oC / 1 min at 68^oC and end with 10 min at 68^oC

SECOND PCR

1. dispense 22.5 ul aliquots of PCR-mix B into thin-walled PCR tubes and add 2.5 ul from the first PCR
2. perform the second PCR with conditions as for the first PCR, but for 23 cycles

Alternative, one-step PCR
Place each filter in a PCR tube containing 50 ul 1x buffer II (Applied Biosystems) and incubate 5 min at 95^oC. Take a 2.5 ul aliqout and perform a SECOND PCR (see above); the number of PCR cycles of the second PCR needs to be adjusted accordingly, usually about 2 additional cycles (depending on the probe set).

PREPARATION CAPILLARY ELECTROPHORESIS FRAGMENT RUN

1. add 9.5 ul formamide and 0.5 ul size standard into each well of a 96 well plate
2. add 2 ul PCR product
3. following addition of the PCR products the plate is vortexed and heated for 5 min at 95^oC
4. cool the plate on ice prior to being placed in the capillary sequencer

Material

• blocker-mix: 20 uM of blocking primers (these are the same as the primers used for amplification, i.e. MAPH-F and MAPH-R)
• capillary sequencer: ABI3700 (Applied Biosystems)
• H₂O:
• formamide: (Applied Biosystems)
• herring sperm DNA: 10 mg/ml
• human Cot-1 DNA: 1.0 mg/ml
• NaH₂PO₄: 1.0 M NaH₂PO₄
• Na₂HPO₄: 1.0 M Na₂HPO₄
• NaOH: 1.0 M NaOH
• PCR-mix A: (per filter)
  • 5 ul 10x buffer II (Applied Biosystems)
  • 4 ul 25mM MgCl₂
  • 1.25 ul 10 mM dNTP
  • 0.25 ul Taq-polymerase (5 U/ul, Applied Biosystems)
  • primers; 0.5 ul MAPH-F primer (20 uM) and 0.5 ul MAPH-R primer (20 uM)
  • 38.5 ul H₂O
• PCR-mix B:
  • 2.5 ul 10x buffer II (Applied Biosystems)
  • 2 ul 25mM MgCl₂
  • 0.625 ul 10 mM dNTP
  • 0.25 ul Taq-polymerase (5 U/ul, Applied Biosystems)
  • primers; 0.25 ul MAPH-F primer (20 uM) and 0.25 ul MAPH-R primer (20 uM)
    NOTE: either the forward or reverse primer should be labeled, not both
  • 16.625 ul H₂O
• pre-hybridisation solution:
  • 280 ul 1.0 M NaH₂PO₄
  • 720 ul 1.0 M Na₂HPO₄
  • 700 ul 20% SDS
  • 276 ul H₂O
  • 20 ul herring sperm DNA
  • 4 ul 0.5 M EDTA (pH8.0)
• primers:
  • MAPH-F (forward primer): 5'-GGCCGCGGGAATTCGATT-3'
  • MAPH-R (reverse primer): 5'-GCCGCGAATTCACTAGTG-3'
• probe-mix: each probe should be present at a concentration of 100 pg/ul (in H₂O). Probes are cloned in pGEM-T easy (Promega) in E.coli XL1-blue.
  NOTE: we have successfully combined up to 50 probes in one hybridisation
• size standard: (Applied Biosystems)
• Stratalinker: UV-cross linker (Stratagene)
• wash solution A:
  • 1 x SSC
  • 1% SDS
• wash solution B:
  • 0.1 x SSC
  • 0.1% SDS

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