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DHPLC analysis of the DMD gene

(last modified on December, 2006)

Contributed by Dick Bennett  -  bennett @ rascal.med.harvard.edu

(full address: Richard R. Bennett, Genetics Division, Children's Hospital, Boston MA, USA)

The data provided here have been published as:

Richard R. Bennett, Johan T. den Dunnen, Kristine F. O'Brien, Basil T. Darras and Louis M. Kunkel (2001). Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing. BMC Genet. 2: 17.

Muscarella et al. (2006) published a modification of the protocol introducing the analysis of 16 couples of amplicons in biplex exons DHPLC runs, making the assay more rapid and less expensive. 


PCR reaction - Abgene Thermo Start AB-0908/b

Ingredient Amount (in ul)
H2O 10.425 17.375 34.75 52.125  55.6 69.5 90.35
10x buffer 1.5 2.5 5.0 7.5  8.0 10.0 13.0
25 mM MgCl (16.7x) 0.9 1.5 3.0 4.5  4.8 6.0 7.8
2.5 mM/each  dNTP (12.5x) 1.2 2.0 4.0 6.0  6.4 8.0 10.4
forward primer (100 ng/ul) 0.3 0.5 1.0 1.5  1.6 2.0 2.6
reverse primer (100 ng/ul) 0.3 0.5 1.0  1.5 1.6 2.0 2.6
DNA-polymerase (5.0 Units/ul) 0.075 0.125  0.25  0.375 0.4 0.5 0.65
template DNA (100 ng/ul) 0.3 0.5 1.0  1.5 1.6 2.0 2.6
TOTAL : 15 25 50 75  80 100 130

All reactions are performed at the annealing temperature (T-anneal) specified for each primer pair (see DHPLC-primers). PCR; 15 min 95oC (enzyme activation), followed by 35 cycles of 5 sec 94oC, 15 sec at specified T-anneal, ramp rate 0.5oC/sec down from 94oC and 30 sec 72oC. Final elongation for 3 min at 72oC. Store at 4oC.

PCR reaction - Qiagen HotStar 203205

Ingredient Amount (in ul)
H2O 8.325  13.875 27.75 41.625  44.4 55.5 72.15
10x buffer 1.5 2.5 5.0  7.5 8.0 10.0 13.0
Q solution (5x) 3.0 5.0  10.0 15.0  16.0 20.0 26.0
2.5 mM/each  dNTP (12.5x) 1.2 2.0 4.0  6.0 6.4 8.0 10.4
forward primer (100 ng/ul) 0.3  0.5 1.0  1.5 1.6 2.0 2.6
reverse primer (100 ng/ul) 0.3  0.5 1.0  1.5 1.6 2.0 2.6
DNA-polymerase (5.0 Units/ul) 0.075  0.125 0.25  0.375 0.4 0.5 0.65
template DNA (100 ng/ul) 0.3  0.5 1.0  1.5 1.6 2.0 2.6
TOTAL : 15  25 50  75 80 100 130

All reactions are performed at the annealing temperature (T-anneal) specified for each primer pair (see DHPLC-primers). PCR; 15 min 95oC (enzyme activation), followed by 35 cycles of 5 sec 94oC, 30 sec at specified T-anneal, ramp rate 0.5oC/sec down from 94oC and 45 sec 72oC. Final elongation for 5 min at 72oC. Store at 4oC.

NOTE 1: for the T-anneal, use the temperature given in the header for each fragment. This will limit the entire set to five groups of T-anneal, i.e. either 50oC, 52oC, 54oC, 56oC or 58oC.

NOTE 2: the Hybaid thermalcycler with "Active Tube Control" was used for testing. Other thermal cyclers and polymerases/ kits will have to be optimized for best T-anneal and other variables such as number of cycles for maximum product with minimum non-specific bands. If you see non-specific bands on agarose or poor WAVE-shapes on the WAVE, try increasing T-anneal and/or decreasing the number of cycles before attempting more drastic measures such as changing dwell times, reagent concentrations, polymerases or primer designs.

NOTE 3: although in some cases (e.g. certain GC rich exons for example) you may get more product, i.e. one to several more milliVolts absorbance, or better WAVE-shape for the same number of cycles using QIAgen Hot Star, you can probably get away with half as much DNA or less as well as shorter dwell times with the ABgene Thermo-Start. You cannot get away with significantly less DNA or shorter dwell times with the Qiagen Hot Star. Also, for most fragments, Q-Solution can be substituted for by water to save Q-solution. A few fragments will work better with Q-solution than without and a few will work better without Q solution than with it. Eventually, these will all be noted in the database as well as whether one enzyme kit seems to work significantly better than the others for a given fragment. Another Polymerase kit that probably worked well with all fragments but has not been tested yet is AGS Gold from Hybaid GmbH. Cat. No. A00797XS. This polymerase allows much shorter dwell times (as low as 2 seconds at all three temperatures—denature, anneal and extend). Also, Qiagen just announced a new high end polymerase called ProofStart. Catalog # 202205 which offers both Proof reading and Hot Start. This may well be the optimum choice, but of course is untested yet and may be more expensive than the others.

NOTE 4: use unpurified PCR product for WAVE-analysis. Be aware that certain buffers/polymerase kits are not allowed on the WAVE. They clog up the column. You will need 5 ul per injection for most fragments. A few which have weaker bands will require 10 ul or more and these will be noted in the database. Plan on a 50oC injection and 3 other temperature injections until you are more familiar with each fragment.

NOTE 5: for the WAVE, for DMD patients (hemizygous) you need to mix half and half an unaffected persons PCR product with your patients PCR product after the PCR’s have been completed separately. Then heat the mix to 95oC for 5 minutes and cool to RT slowly over 45 min in a thermalcycler.

NOTE 6: purify PCR products for sequencing with Qiagen PCR kit. You probably need about 40 to 50 ul.   Therefore, you need the following amounts of PCR product: For the WAVE, for the patient, for most fragments, you need 4 x 5u l plus 10 ul for the pot of the mix of affected and unaffected. i.e 15 ul affected plus 15 ul unaffected. You also need for the wave, 30 ul unaffected to run separately on the WAVE as a control so that you can compare resulting wave forms. You should also do a 15 or 25 ul negative (water) control. So if you are just screening with the WAVE, you usually (depending on the number of injections and the strength of the PCR product) just need 25 ul PCR for the patient and a 50 ul PCR for the unaffected plus a 15 ul or 25 ul negative control. However, add 10 ul to the patient and the unaffected PCR’s if you are planning to run them on an agarose gel, and add 50 ul to the patient PCR if planning to sequence.

NOTE 7: for the WAVE, use a separate line in the sample sheet for each temperature, as opposed to using the multiple injection feature. Just use the temperatures in parenthesis for the first pass which will ferret out most exonic mutations. If no mutations are found for a given patient, you must decide whether to make a second pass with temperatures outside the parenthesis to ferret out sticky exonic and/or intronic mutations/polymorphisms or to go directly to direct sequencing for all exons. Also for the WAVE, use 7.6 seconds run time (4.5 seconds gradient time) with fast clean option ( if you have the accelerator option) and accept the default start and stop acetonitrile gradients (i.e. what they call the “virtual method”). There is no need to name or save the method or even to look at the gradient page.

NOTE 8: be sure your WAVE has an oven controller capable of controlling oven temperature in 0.1oC increments. Older units only control to 1.0oC increments, but and upgrade option is available.

NOTE 9: it is critically important that the size and mutation standards be run at a bare minimum of once per week ( preferably once a day). The report should be compared against the report of the first (or best) run of the two standards which is usually done on the day of installation. This is to insure that the column is still separating well.   Patient fragments whose wave shape varies more than slightly from the unaffected control fragment at any temperature of course must go to sequencing for verification and detailed analysis.

Sequence analysis

Purify PCR products with Qiagen PCR kit. Sequences are run on the ABI373 flourescent sequencer. Reactions for the sequencer contain per reaction:

Ingredient Amount (in ul)
H2O 8.0
primer (20 ng/ul) 4.0
template DNA (1.5 ng/ul per 100 bp)  4.0
TOTAL : 20.0

Template DNA (1.5 ng/ul per 100 bp), i.e. use 12 ng total for fragments up to 200 bp; 18 ng for fragments between 200 and 400 bp and 24 ng total for fragments larger than 400 bp.

Cycle extension progam in the ABI9600 thermal cycler is 4 min 95oC followed by 25 cycles of 95oC for 12 sec, 49oC for 6 sec and 60oC for 4 min. Purify reactions with BIO-RAD SEQueaky Kleen 96 well kit.

WAVE analysis

For conditions of WAVE analysis see DHPLC-primers and WAVE conditions. It is VITALLY important once you have found a causative mutation in one fragment of a gene to continue the screening until all fragments have been screened because you would not want to be responsible for telling someone who has a family member with the mutation that they are clean only to discover later that there was another causative mutation in another fragment of the gene.

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