Abstract

Depending on the desired resolution and sequencing scale, different restriction enzymes can be used for genomic DNA digestion. PstI is used here as an example. Among the 6-base restriction enzymes, PstI has the highest restriction frequency in the human genome sequences (Chen J., et al. 2008. Scanning the human genome at kilobase resolution. Genome Research 18(5):751-762).

Procedure

Restriction digestion of genomic DNA

1. Digest genomic DNA with PstI.

Attaching PstI-MmeI-Solexa adaptor to genomic DNA fragments

test The PstI-MmeI-Solexa adaptor (synthesized, gel-purified, and double strand−formed by IDT) (Figure 2) contains Solexa adaptor A and B sequences and two MmeI sites closed to the mutated PstI ends. A PstI site is added between Solexa adaptor A and B. It can be used to remove the artificially ligated adaptor−adaptor complex.

1. Ligation

test

2. Incubate at 4°C overnight or room temperature for 3 h.

3. Extract, precipitate, and resuspend DNA in 30 µL ddH₂O.

Digestion DNA fragments with PstI to keep single adaptor A and adaptor B

test

1. Incubate at 37°C for at least 1 h.

test 2. Evaluate digestion efficiency by loading 3 µL digested DNA on a 1% agarose gel, and load undigested adaptor-ligated genomic DNA at the same time (Figure 3).

3. Recover digested products from the gel that are over 76 bp in length with QIAquick Gel Extraction Kit (Qiagen) to remove the digested adaptors, and resuspend ligation products in 90 µL ddH₂O. Check 5 µL recovered DNA on a 1% agarose gel.

Re-circularization of the purified DNA

test

1. Incubate at 4°C overnight or room temperature for 3 h.

2. Extract, precipitate, and resuspend DNA in 30 µL ddH₂O.

MmeI digestion of circulated DNA

test

1. Incubate at 37°C for 1 h.

test 2. Evaluate digestion efficiency by loading 5 µL digested DNA on 2.5% agarose gel, using MmeI-digested pGEM as positive control (Figure 4).

3. Recover 116 bp MmeI-digested products with QIAquick Gel Extraction Kit (Qiagen) and resuspend DNA in 30 µL ddH₂O.

Blunt the Tag-Adaptor-Tag fragments

test

1. Incubate at 12°C for 15 min, add 1 µL of 0.5 M PH7.5 EDTA, and inactivate at 75°C for 20 min.

2. Extract, precipitate, and resuspend DNA in 20 µL ddH₂O.

Form ditags by Tag-Adaptor-Tag fragments ligation

test

1. Incubate at 15°C for 4–18 h.

PCR amplification of ditags with Solexa primers

Solexa PCR forward primer:
5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′

Solexa PCR reverse primer:
5′-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3′

Sequencing Primer1:
5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′

Sequencing Primer2:
5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3′

1. Pre-PCR test

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PCR conditions: 98°C for 30 s, 35 cycles of 98°C for 5 s, 60°C for 5 s; and 72°C for 20 s; 72°C for 1 min, 4°C hold. Use adaptor-ligation products as positive control.

2. Check 5 µL on a 2.5% agarose gel to ensure positive amplification of the 160-bp fragments.

3. Large-scale PCR (20 wells per sample) test

PCR conditions: 98°C for 30 s, 35 cycles of 98°C for 5 s, 60°C for 5 s; and 72°C for 20 s, 72°C for 1 min, 4°C hold.

4. Combine all PCR products into two 1.6-mL Eppendorf tubes (200 µL/tube).

5. Extract, precipitate, and resuspend DNA in 100 µL ddH₂O.

6. Load the entire DNA on a 2.5% agarose gel, excise, and purify the ditag fragments (about 160 bp) that contain ditags and Solexa PCR primers using QIAquick Gel Extraction Kit (Qiagen); resuspend in 50 µL Buffer EB (Qiagen).

7. Check the purified DNA by gel and quantify by optical density (OD). The purified ditag DNA templates are ready for Solexa sequencing reaction. 500–1000 ng will be sufficient for Solexa sequencing collection.

Reagents

• NEBuffer 2 [Cat. no. B7002S; New England BioLabs (NEB), Ipswich, MA USA]

• NEBuffer 3 (Cat. no. B7003S; NEB)

• NEBuffer 4 (Cat. no. B7004S; NEB)

• BSA (Cat. no. B9001S; NEB)

• PstI (Cat. no. R0140S; NEB)

• S-adenosylmethionine (SAM) (Cat. no. B9003S; NEB)

• MmeI (Cat. no. R0637L; NEB)

• T4 DNA Polymerase (Cat. no. M0203L; NEB)

• Phire Hot Start DNA Polymerase (Cat. no. F-120S; NEB)

• 5× Phire Reaction Buffer (Cat. no. F-524S; NEB)

• Glycogen (Cat. no. 10901393001; Roche Diagnostics Corporation, Indianapolis, IN USA )

• Alcohol, ethyl (Cat. no. AB00138; American Bioanalytical, Natick, MA, USA)

• T4 DNA Ligase (Cat. no. M1804; Promega U.S., Madison, WI, USA)

• 10× T4 DNA Ligase Buffer (Cat. no. C1263; Promega U.S.)

• 10 mM dNTP Mix (Cat. no. U1515; Promega U.S.)

• Agarose, LE, Analytical Grade (Cat. no. V3121; Promega U.S.)

• QIAquick Gel Extraction Kit (Cat. no. 28704; Qiagen Inc., Valencia, CA, USA)

• PstI-MmeI-Solexa Adaptor [Integrated DNA Technologies Inc. (IDT), Skokie, IL, USA]

• Solexa forward/reverse primers (IDT)

• Phenol:chloroform (Cat. no. 0883; Amresco Inc., Solon, OH, USA)

• Ammonium acetate (NH4OAc) (Cat. no. 0103; Amresco Inc.)

• EDTA (Cat. no. 0105; Amresco Inc.)

• ddH₂O (made in-house)

Equipment

• 1.6-mL Ultra Clear Graduated Micro Tube (Cat. no. 3445; Biotix, Inc., San Diego, CA, USA)

• GeneAmp PCR System 9700 (Cat. no. N8050200; Applied Biosystems Inc., Foster City, CA, USA)

• Centrifuge (Cat. no. 5415C; Eppendorf North America, Westbury, NY, USA)

• BioPhotometer (Cat. no. RS232C; Eppendorf North America)

• Digital Dry Bath (Cat. no. D1100; Labnet International, Inc., Woodbridge, NJ USA)

• Gel XL Ultra (Cat. no. E0145A; Labnet International, Inc.)

Address correspondence to San Ming Wang, Center for Functional Genomics, ENH Research Institute, 1001 University Place, Evanston, IL, 60201, USA. email: [email protected]