Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

The overall goal of this procedure is to determine the presence or absence of a panel of specific nucleic acid sequences previously amplified in a multiplex PCR reaction. This is accomplished by first fixing amine labeled DNA probes to a reusable nylon membrane. Biotin labeled PCR products are then hybridized to the membrane bound probes and strep TTR in peroxidase.

Conjugate is added to the bound PCR products. Next, the bound PCR products are detected by adding chemiluminescence detection reagents, and exposing the membrane to high sensitivity x-ray film. Finally, the membrane is washed to remove the hybridized PCR products, allowing it to be reused for future assays.

Ultimately, results can be obtained that show hybridization of PCR products to the membrane membrane-bound probes on the x-ray film. The main advantages of this technique over existing methods such as multiplex PCR, with gel electrophoresis real-time PCR or microarray, is that it's high throughput, relatively rapid, does not require any specialized equipment, and most importantly is inexpensive. Before beginning, prepare the following solutions according to the written portion of this protocol.

To prepare the membrane, preheat the oven to 60 degrees Celsius. Clean the mini blotter with 70%ethanol, dilute the oligonucleotide probes in 0.5 molar sodium bicarbonate to a final concentration of two picomoles per microliter and a volume of 200 microliters. Cut the nylon membrane to 15 by 15 centimeters using a pencil and ruler mark off a 0.5 centimeter space across the top of the membrane, and write the details of the experiment there.

Seal the membrane in a plastic bag with 20 milliliters of freshly made 16%EDAC solution and rock it room temperature for 10 minutes. Wash the membrane in deionized water for 30 seconds. Next, place the membrane in a mini blotter with channels running across the membrane.

Then put the support cushion in place and close the blotter. Thoroughly aspirate any fluid from the channels. Fill lanes one and 45 with 150 microliters of 0.5 molar sodium bicarbonate.

Use 150 microliters of each probe solution to fill lanes two to 44 in sequence being careful to avoid air bubbles. If an air bubble does appear in the channel, keeping the pipette in place rapidly aspirate the solution to allow the air bubble to float to the top of the pipette tip, then retry. Incubate at room temperature for five minutes.

Aspirate the probe solutions. Remove the membrane and wash it in 250 milliliters of 0.1 molar sodium hydroxide at room temperature for nine minutes. Wash the membrane in 250 milliliters of two times SSPE for 30 seconds.

Then wash it in 250 milliliters of prewarm two times SSPE 0.1%SDS in the oven at 60 degrees Celsius for five minutes, if not immediately being used for hybridization. Wash the membrane in 240 milliliters of 20 millimolar EDTA at room temperature for 20 minutes. Then sell the membrane in a plastic bag with the remaining 10 milliliters of 20 millimolar EDTA and store at four degrees Celsius.

Finally, wash the mini blosser with pyro neck detergent and a brush rinse and allow it to dry multiplex. PCR Amplification of the targets of interest is not described in this demonstration, but must be performed prior to pro hybridization. Before beginning the hybridization procedure, prepare the following solutions according to the written portion of this protocol.

Turn on one oven and set to 60 degrees Celsius and another oven to 42 degrees Celsius. Bring the water to a boil in a large beaker on a hot plate. Clean the mini blotter with 70%ethanol.

Next aliquot 10 milliliters of two times SSPE 0.1%SDS into a small container. Then add 20 microliters of each PCR product to 150 microliters of two times SSPE 0.1%SDS in numbered tubes. Boil the PCR products for 10 minutes at 100 degrees Celsius using styrofoam holders place on ice immediately for at least five minutes.

Wash the membrane in the remaining 240 milliliters of two times SSPE 0.1%SDS in the 60 degree Celsius oven for five minutes. Place the membrane in the mini blotter with backing cushion. Orient it so that the channels run vertically.

Suction the excess liquid from the mini blotter channels. Add the remaining 150 microliters of two times SSPE 0.1%SDS to the first and last channels. Add boiled PCR products to channels two to 44 in sequence being careful to avoid air bubbles.

Place the mini blotter flat in the 60 degree celsius oven for one hour to allow hybridization to take place. Spray the liquid from each channel. Then remove the membrane from the mini blotter.

Washed twice in pre-warned 250 milliliters of two times SSPE, 0.5%SDS in the 60 degree Celsius oven for 10 minutes. Moisten the nylon backing with two times SSPE 0.5%SDS at 42 degrees Celsius and use it to roll up the membrane. Place the rolled up membrane into a roller tube and unfer to ensure the membrane abuts the interior surface.

Add three microliters of strept AD in peroxidase conjugate to 15 milliliters of two times SSPE, 0.5%SDS at 42 degrees Celsius and add to the tube. Screw the cap on tightly and incubate in the roller oven at 42 degrees Celsius for 60 minutes. Ensure the direction of the roll is such that the membrane does not tighten.

Check periodically for leaks. Wash the mini blotter in pyro neck detergent and water rinse and allow it to dry. Remove the membrane from the roller tube wash twice in the remaining two times SSPE 0.5%SDS at 42 degrees Celsius for 10 minutes.

Then wash twice in two times SSPE at room temperature for five minutes. Turn both ovens up to 80 degrees Celsius and place 500 milliliters of 1%SDS in one oven to prewarm. Make up the ECL solution and incubate the membrane in ECL detection solution for two minutes.

Then expose the membrane to film and develop. Repeat the exposure with longer or shorter times if required to regenerate the membrane. Wash it in 250 milliliters of 1%SDS at 80 degrees Celsius for 30 minutes twice.

Then Washington 240 milliliters of 20 millimolar EDTA at room temperature for 15 minutes. Seal the membrane in a plastic bag with 10 milliliters of 20 millimolar EDTA and refrigerator at four degrees Celsius for future reuse. This figure shows the results from a multiplex PCR reverse line blot hybridization assay Samples one through six represent positive controls between them.

There is at least one positive probe signal for each of the 43 probes. Each target sequence A through U is detected with two different probes to maximize specificity probes. A one and a two represents species specific probes, which are expected to be positive for each sample and assist with orienting the film probe.

A one is repeated at the bottom of the membrane. Samples seven and eight are negative controls. Note that probes Q1, Q2, T one and T two have signal in the negative controls indicating likely nonspecific binding of primers or primer dimer.

Product redesign of these probes or primers would be required probes, C one, D one and F1 show streaking across the membrane, likely due to some non-specific uptake of the strep TD in peroxidase, conjugate, or chemiluminescent substrate. However, this is easily distinguished from true positive probes. Signals probes D one and D two have relatively weak signals compared to the other probes.

This may be due to larger amplicons leading to less efficient amplification. Finally, probe J two is negative in several samples where probe J one is positive. This most likely represents a mutation in the J two binding site for these samples.

Once mastered this technique from DNA extraction through PCR Pro hybridization, result interpretation and regeneration of the membrane can all be done in less than 10 hours.