Protocol 61 Target preparation


Reagents for genomic DNA isolation.

Depending on the sample source, type and quantity, the genomic DNA isolation method can vary. Select the one optimal for each sample.

PCR reagents:

Hotstar Taq master mix (Qiagen. Cat. no. 203445)

Primers: 15 mM forward and reverse primers in DEPC-treated water

Genomic DNA samples

PCR product quantification and visualization:

Agilent DNA12000 labchip kit (Agilent. Cat. no. 5064-8231)

PCR product precipitation:

7.5 M ammonium acetate 100% ethanol

In vitro transcription reagents:

T7 Megascript kit (Ambion, Inc. Austin, TX. Cat. no. 1334)

Target labeling reagents and material:

1mM Fluorolink Cy3-dUTP and Fluorolink Cy5-dUTP (Amersham Biosciences Corp. Piscataway, NJ. Cat. no. PA53022 and PA55022)

Superscript II RNaseH- (with 5 x first strand buffer and 50 mM DTT) (Invitrogen Corp. Carlsbad, CA. Cat. no. 18064-07)

50 mM EDTA

1 M NaOH

Microbiospin 6 columns (Bio-Rad. Cat. no. 732-6222) pd(N)6 (Boerhinger Mannheim. Cat. no. 1034731) 1 x TE

Microcon YM-30 column (Millipore. Cat. no. 42410).

Hybridization reagents:

50 x Denhardt's blocking solution (Sigma. Cat. no. 2532) Poly dA 40-60 (8 mg ml-1) ( Pharmacia. Cat. no. 27-7988-01) Human Cot I DNA (1 mg ml-1) (Invitrogen. Cat. no. 15279-011) 20 x SSC 10% SDS

Hybridization chambers (Corning. Cat. no. 2551)

Array scanner:

GenePix 4000B scanner (Axon Instrument)


PCR reaction

PCR setting for generation of SNP-typing target. PCR reaction mixture: 1 |l of 5' T7-primer (15 |M)

10.5 |l of genomic DNA (containing approximately 50-100 ng of genomic DNA)

12.5 |l of hot start mixture

Total volume 25 |l

PCR profile: 95°C for 10min

5 cycles

72°C for 3min

20 cycles

72°C for 3min

9 cycles

Run Agilent DNA chip (Figure 6.1).

10380 bp 7000 bp 5000 bp

3000 bp

2000 bp 1500 bp

1000 bp

700 bp

500 bp

300 bp

100 bp 50 bp

10380 bp 7000 bp 5000 bp

3000 bp

2000 bp 1500 bp

1000 bp

700 bp

500 bp

300 bp

100 bp 50 bp

Figure 6.1.

Agilent Bioanalyzer DNA7500 chip analysis. A 2000-bp genomic DNA fragment of HLA A locus spanning exon 1 to exon 6 was PCR amplified using a T7-gene-specific primer pair. Lane 1, molecular weight marker; lane 2-5, genomic DNA fragments amplified from CL013, CL033, CL018 and CL096 cell line genomic DNA.

PCR product precipitation

Add 12.5 |l of 7.5 M ammonium acetate to the PCR product (25|l volume).

Centrifuge at 13 000 g for 20 min at room temperature to avoid co-precipitation of primers.

Wash with 500 |l of 100% EtOH twice.

Dry pellet completely and then re-suspend in 10 |l of DEPC-treated water.

Check DNA amount by either Agilent Bioanalyzer or spectrophotometer.

In vitro transcription using T7 Megascript Kit

2 |l reaction buffer

2 |l enzyme mix (RNase inhibitor and T7 phage RNA polymerase) 1 |g of PCR amplified DNA in 8 |l volume Incubation at 37°C for 6 h.

Purification of amplified RNA

Any manufactured RNA isolation kit can be applied. A monophasic reagent such as TRIzol reagent from Invitrogen (cat. no. 15596026) is exemplified here based on the efficient recovery of aRNA. Other methods for RNA isolation can also be employed.

1. Add 1 ml of TRIzol solution to the transcription reaction. Mix the reagents well by pipetting or gentle vortexing.

2. Add 200 |l chloroform per ml of TRIzol solution. Mix the reagents by inverting the tube for 15 s. Allow the tube to stand at room temperature for 1-2 min.

3. Centrifuge the tube at 10 000 g for 15 min at 4°C.

4. Transfer the aqueous phase to a fresh tube and add 500 |l of isopropanol per ml TRIzol reagent.

5. Store the sample at room temperature for 5 min and then centrifuge at 13 000 g for 20 min.

6. Wash the pellet twice with 1 ml 70% EtOH.

7. Allow the pellet to dry in air and then dissolve it in 30 |l of DEPC H2O.

8. Measure the quantity of RNA using the Agilent Bioanalyzer RNA 6000 chip (Figure 6.2).

Target labeling by reverse transcription

4 |l First strand buffer

2 |il 10 x lowT-dNTP (5 mM A, C and GTP, 2 mM dTTP) 2 |l Cy-dUTP (1 mM Cy3 or Cy5)

3 |g amplified RNA in 8 |l DEPC H2O Mix well and heat to 70°C for 3 min then cool down to 42°C.

Add 1 |l SSII. Incubate for 30 min at 42°C and add another 1 |l SSII for 40 min at 42°C. Add 2.5 |l 500 mM EDTA and heat to 65°C for 1min. Add 5 |l 1 M NaOH and incubate at 65°C for 15 min to hydrolyze the RNA. Add 12.5 |l 1 M Tris immediately to neutralize the pH. Bring the volume to 70 |l by adding 35 |l of 1 x TE.

Note: The amounts of aRNA used for labeling depend on the size of the array. If the array

6000 nt 4000 nt

2000 nt

1000 nt 500 nt 200 nt

6000 nt 4000 nt

2000 nt

1000 nt 500 nt 200 nt

Figure 6.2.

Agilent Bioanalyzer RNA 6000 chip analysis. A 2000-nt RNA fragment corresponding to the HLA A locus from exon 1 to exon 6 was amplified using the T7 Megascript kit. Lane 1, RNA ladder; lane 2-4, amplified RNA using CL013, CL033, CL018 and CL096 cell line genomic DNA fragment as templates.

contains 2000-8000 oligo probes, 3 Mg aRNA will be sufficient while a larger chip such as one containing 16-20 k oligo probes will need 6 Mg of aRNA. The labeling reaction components do not need to be changed.

Target clean up: Prepare a Bio-6 column and apply the target solution through it according to the manufacturer's instructions. Collect flow through and add 250 Ml 1 x TE to it. Concentrate target to around 20 Ml using a Microcon YM-30 column.

Hybridization: Combine Cy3-labeled reference sample and Cy5-labeled test target

(adjust the color to purple in order to balance the amount of test and reference samples) and then completely dry the sample using a Speedvac. Re-suspend the pellet in 25 Ml volume by adding 1 Ml 50 x Denhardt's blocking solution, 1 Ml poly dA (8 Mg/Ml), 1 Ml yeast tRNA (4 mg/ml), 10 Ml Human Cot I DNA (1 mg/ml), 3 Ml 20 x SSC, 0.6 Ml of 10% SDS and 8.4 Ml of DEPC-treated water. Heat the solution for 2 min at 99°C and apply this target mixture to the slide, add a coverslip, place the slide into a humidified hybridization chamber (Corning. Cat. no. 2551), and hybridize at 45°C overnight.

Slide washing: 1. Wash with 2 x SSC + 0.1% SDS to get rid of the cover slip.

5. Centrifuge slide at 80-100 g for 3 min (the slide can be put in a slide rack on a microplate carrier or in a 50-ml conical tube and centrifuged in a swinging-bucket rotor).

Scan slide.

From gene chips to disease chips - new approach in molecular diagnosis of eye diseases

Rando Allikmets and Jana Zernant

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