• CamSysBiol
• Proteomics
• FlyMine
• FlyBase
• INDAC
• DrosDel
• Affymetrix

Ligation-mediated PCR (LM-PCR)

Overview

Following ChIP, there is insufficient genomic DNA for labelling and hybridisation so we amplify with LM-PCR. We first repair the genomic DNA to make the amplicons blunt ended then ligate on the linkers. Afterwards, ligation-mediated PCR is performed using a single primer to ensure that the amplification is 'linear'. Random prime labelling is then performed using one of two commercial kits. Pre-immune and immune ChIP genomic DNA are then purified and mixed before hybridisation.

Warning:

The following procedures require pure DNA and the Qiagen column sometimes leaves a small white salt pellet in the microfuge tubes. Whenever a qiagen column is used, care must be taken to check whether a pellet is present. Since the pellets can rapidly dissolve, these must be removed immediately after centrifugation.

Protocol

Reparing the sheared genomic DNA after ChIP

Prepare the following reaction mix and incubate for 5 minutes at 37 °C

• 15 to 30 µl ChIP genomic DNA
• 1 µl T4 DNA polymerase (Promega; Cat. No. M4211)
• 5 µl 10X T4 polymerase buffer
• 2.5 µl 2 mM dNTP
• Make up to 50 µl with water

DNA is purified with a Qiagen MinElute PCR purification Kit (Cat. No.28004), as per the manufacturers protocol. Elute the repaired ChIP genomic DNA with a volume of water that is equal to the starting amount of ChIP genomic DN, i.e., 15 to 30 µl. Remember to remove the white pellet, if present.

Linker preparation

Linker-1 (100 pmol/µl) 5'-AGA AGC TTG AAT TCG AGC AGT CAG-3'
Linker-2 (100 pmol/µl) 5'-CTG CTC GAA TTC AAG CTT CT-3'

Mix 2.5 µl of each linker with 45 µl water

• Incubate for 2 minutes at 94 °C
• Incubate for 5 minutes at 70 °C (To remove secondary structures)
• Incubate for 5 minutes at 55 °C (annealing)

Slowly cool down to room temperature to obtain 50 µl of 5 µM blunt linker. Add 200 µl water to get 1 µM linker and store at -20 °C.

Ligation of repaired ChIP genomic DNA to linker

15 µl of ligation reaction was prepared:

• 10.5 µl repaired ChIP genomic DNA
• 1 µl 1 µM linker
• 1 µl 5 U/µl T4 DNA ligase (Invitrogen; Cat. No. 15224-041)
• 2 µl 5X ligase buffer
• 0.5 µl Water

Mix and incubate overnight at 4 °C

Ligation-mediated PCR (LM-PCR)

Clean the overnight ligation reactions with the Qiagen MinElute PCR purification Kit (Cat. No.28004), as per the manufacturers protocol. Elute the ChIP genomic DNA with 17 µl water. Remember to remove the white pellet, if present. Keep the DNA on ice and prepare the following.

Reaction mix

• 15 µl Ligated DNA
• 10 µl 10X buffer 4 (no Mg²⁺) (ABgene)
• 8 µl 25 mM Mg²⁺
• 8 µl 2 mM dNTPs
• 1 µl Taq polymerase (ABgene; Cat. No. AB-0192)
• 1 µl 100 pmol/µl linker-2
• 57 µl water

PCR cycle

1. 55 °C for 2 minutes
2. 72 °C for 5 minutes
3. 94 °C for 5 minutes
4. 94 °C for 1 minute
5. 55 °C for 1 minute
6. 72 °C for 1 minute
7. Repeat cycles 4 to 6, 24 times
8. 72 °C for 5 minutes
9. 4 °C forever

DNA is purified with a Qiagen MinElute PCR purification Kit (Cat. No. 28004), as per the manufacturers protocol. Elute LM-PCR ChIP genomic DNA with 25 µl water. Remember to remove the white pellet, if present.

Random priming and dye labelling

Measure sample concentrations using the Nanodrop, as per the RNA quality control protocol for gene expression, i.e., follow this standard operating procedure but set the Nanodrop to assay DNA. We label 100 ng template using the BioPrime array CGH genomic labelling system (Invitrogen; Cat. No.18095-011).

BioPrime array CGH genomic labelling system

Make the following reaction mix and incubate for 5 minutes at 95 °C.

• 25 µl Purified PCR product (100 ng DNA, plus water to 25 µl)
• 20 µl 2.5X Random primer mix

Immediately cool for 5 minutes on ice. Then, add the following reagents, mix them together and incubate for 2-3 hours at 37 °C

• 1 µl Exo-Klenow Fragment
• 5 µl 10x dCTP or dUTP mix
• 3 µl Cy3 or Cy5

Stop reaction with 5 µl of stop buffer. 3 µl of the reaction is then checked by agarose gel electrophoresis. You should see a well defined smear between 200 to 600bp.

Probe clean-up

1. Add 45 µl TE, PH 8.0 to the labelling mixtures.
2. Add 400 µl Purification buffer A and vortex for 30 seconds.
3. Place the purification column in a 2 ml collection tube, load labelling mixture.
4. Centrifuge at 11,000 x g for 1 minute at room temperature and discard the flow-through.
5. Add 600 µl of Purification buffer B, centrifuge at 11,000 x g for 1 minute at room temperature and discard the flow-through.
6. Add 200 µl of Purification buffer B, centrifuge at 11,000 x g for 1 minute at room temperature and discard the flow-through.
7. Place the collection tube in a new sterile microfuge tube and add 50 µl sterile water. Incubate at room temperature for 1 minute.
8. Centrifuge at 11,000 x g for 1 minute at room temperature. The flow-through contains your purified labelling mixture.
9. Measure the nucleic acid concentration and dye-incorporation with the Nanodrop ND-1000 spectrophotometer. Ensure samples and matched in concentration.
10. The two samples (e.g. pre-immune and immune) can now be combined together for hybridisation to a microarray. The combined samples are reduced in volume to between 2-10 µl in a SpeedVac at medium heat and the blocking agent, sonicated salmon sperm DNA (2 µl of 10 mg/ml), is added. This material should now be used immediately to prevent any decay. Refer to the appropriate hybridisation protocol.

Version 1.2. R. Auburn. (01-09-2006)