Human and Clinical Genetics (LUMC) - LabJ_Protocols©
MAPH - Multiplex Amplifiable Probe Hybridisation
(Stefan White/Margot Kalf; last modified October 4, 2002)
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.
- 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
- 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
- when all the DNA has been applied to the filters, allow to dry and wrap up in the cling
film
- crosslink the DNA to the filters by UV irradiation (50 mJ, both sides, Stratalinker)
PRE-HYBRIDISATION AND HYBRIDISATION
- prepare fresh pre-hybridisation solution
- pre-hybridise the filters together in an Eppendorf tube containing 1 ml
pre-hybridisation solution and incubate 2 h to O/N at 60oC
HYBRIDISATION
- prepare 300 ul pre-hybridisation solution, add 3 ul human Cot-1 DNA and boil 2 min in an
Eppendorf tube
- 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 60oC
- prepare hybridisation solution;
- 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
- add 2 ul 1.0 M NaOH, incubate 1 min at 37oC and place on ice
- 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
- incubate O/N at 60oC
POST-HYBRIDISATION WASHES AND PCR
- transfer the filters to a 50 ml tube containing 25 ml pre-warmed (60oC) wash
solution A
- wash for a total of 45-60 min;
- 5 times at 60oC in 25 ml pre-warmed (60oC) wash solution A
- 5 times at 60oC in 25 ml pre-warmed (60oC) wash solution B
- 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.
- 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
- perform the first PCR; 5 min at 94oC, 5 cycles of 45sec at 94oC /
1 min at 57oC / 1 min at 68oC and end with 10 min at 68oC
SECOND PCR
- dispense 22.5 ul aliquots of PCR-mix B into thin-walled PCR tubes and add
2.5 ul from the
first PCR
- 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
95oC. 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
- add 9.5 ul formamide and 0.5 ul size standard into each well of a 96 well plate
- add 2 ul PCR product
- following addition of the PCR products the plate is vortexed and heated for 5 min at 95oC
- 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:
- wash solution B:
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