Published in association with Cold Spring Harbor Laboratory Press

Direct genomic selection

Stavros Bashiardes, Rose Veile, Cynthia Helms, Elaine R Mardis, Anne M Bowcock & Michael Lovett

Department of Genetics, Washington University School of Medicine, Box 8232, 4566 Scott Avenue, St. Louis, Missouri 63110, USA.

Searching for genetic variants and mutations that underlie human diseases, both simple and complex, presents particular challenges. In the case of complex diseases, these searches generally result in a single nucleotide polymorphism (SNP), or set of SNPs, associated with disease risk. Frequently, these SNPs lie outside the gene coding regions^{1, 2}. One is thus left in a quandary: do the detected SNPs represent the only genetic variation in the region or are there additional variants that might show even higher associations with disease risk? In the case of cancer, identification of mutations in tumor suppressor genes has also proved to be an arduous and frequently fruitless task³. The problem also arises in mouse genetics where mutational screens—for example, using ethylnitrosourea (ENU)—frequently require resequencing of large genomic regions to find a single base change⁴. The problem devolves to one of resequencing a large region of genomic DNA, usually of >100 kilobases (kb), from affected individuals or tissue samples to identify all sequence variants. Here, we describe modifications to direct selection^{5, 6} that allow for the rapid and efficient discovery of new polymorphisms and mutations in large genomic regions. Biotinylated bacterial artificial chromosome (BAC) DNAs are used in two rounds of hybridization selection with a target of total genomic DNA, and the selected sequences are amplified by the polymerase chain reaction (PCR) (Fig. 1). The procedure results in enrichments of 10,000-fold, in which 50% of the resulting sequence-ready clones are from the targeted region (Box 1).

Figure 1. A flow diagram of the direct genomic selection process. Specific steps are discussed in the text. We illustrate the proof of concept of the method, also described in Box 1, using a 150-kb BAC from human chromosome 17. Full Figure and legend (46K)

JUMP TO STEPS
	Preparation of the genomic DNA and double-stranded linkers
	Ligation of linkers to genomic DNA fragments
	Biotinylation and blocking of the BAC DNA
	Primary selection and capture of hybrids
	Amplification of the primary selected DNA
	Further enrichment by secondary selection
	Amplification of secondary selected material
	EcoRI digestion and purification of the amplified products

Reagents
Genomic DNA, pooled isolates from individual patients or tissue samples
Restriction enzymes Sau3AI (Promega), HinfI (Invitrogen) and EcoRI (Invitrogen)
DNA polymerases: T4 DNA polymerase (Invitrogen), PfuUltra HF DNA polymerase (Stratagene)
5 T4 DNA polymerase reaction mix: 5 T4 DNA polymerase buffer, 1 mM each dNTP
Oligonucleotide 3: 5'-CTCGAGAATTCTGGATCCTC-3', Oligonucleotide 4: 5'-GAGGATCCAGAATTCTCGAGTT-3'
10 annealing buffer: 100 mM Tris-HCl (pH 7.5), 1 M NaCl, 10 mM EDTA
T4 DNA ligase (Roche)
QIAquick PCR purification kit (Qiagen)
Purified BAC DNA, from clone encoding region of interest
Biotin-16-dUTP (Enzo Biochemicals)
10 nick translation buffer: 500 mM Tris-HCl (pH 7.5), 100 mM MgCl₂, 50 mM DTT, 400 M each dNTP
[-³²P]dCTP (3,000 Ci/mmol) (Amersham)
DNA polymerase/DNase I (Roche)
Cot-1 DNA (Invitrogen)
2 hybridization buffer: 1.5 M NaCl, 40 mM sodium phosphate buffer (pH 7.2), 10 mM EDTA (pH 8), 10 Denhardt's, 0.2% SDS
Streptavidin-coated paramagnetic beads and magnetic bead separator (Dynabeads M-280 Streptavidin)(Dynal)
Streptavidin bead binding buffer: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8) and 1 M NaCl
dNTP solution (25 mM each) (Invitrogen)
p-AMP1 plasmid DNA (Invitrogen)
DH10B electrocompetent Escherichia coli (Invitrogen)

Preparation of the genomic DNA and double-stranded linkers
1. Set up two reactions for digestion of genomic DNA:

Incubate the two reactions at 37 °C for 3 h. Extract the reactions with phenol/chloroform (1:1, v/v), recover the DNA by precipitation with ethanol and dissolve the DNA pellets in 50 l of water. Set aside 3-l aliquots for use as controls below.
This combination of enzymes is used to compensate for fragment length representational differences that might be introduced by digestion with a single enzyme.
 Critical step

2. Set up two reactions to fill in the overhang ends of the DNA digestion products:

Incubate the reactions at 11 °C for 30 min. Combine the two reactions, extract with phenol/chloroform and recover the DNA by ethanol precipitation. Dissolve the pellet in 10 l water (to give a final concentration of 2 g/l).
 Troubleshooting

3. Anneal oligonucleotides 3 and 4 to create a double-stranded linker, by mixing the following:

Heat the reaction at 65 °C for 10 min; then allow it to cool at 15−25 °C for 2 h.
The oligonucleotides in this protocol are designated 3 and 4 to distinguish them from oligonucleotides 1 and 2 used in the related protocol for direct selection of cDNAs⁶.

4. Purify the double-stranded linker by column chromatography through a Sephadex G-50 spin column; then concentrate the purified linker solution by lyophilization to a concentration of 2 g/l.

Ligation of linkers to genomic DNA fragments
5. Set up the following reaction to ligate the linkers to genomic DNA fragments
Incubate the reaction at 14 °C overnight.

 Troubleshooting

6. Adjust the reaction volume to 500 l with water and purify the ligated genomic DNA using a QIAquick PCR purification kit. Store the purified DNA at a concentration of 1 g/l.

Biotinylation and blocking of the BAC DNA
7. To incorporate biotinylated residues into the BAC DNA, prepare the following nick translation reaction:

Incubate the reaction at 4 °C for 2 h.
The isotope is included as a tracer to confirm that the biotinylation reaction has proceeded efficiently and to confirm binding of the BAC DNA to streptavidin-coated magnetic beads.
 Critical step

8. Purify the biotinylated products by column chromatography through a Sephadex G-50 spin column and lyophilize the resulting solution to dryness.

9. Dissolve the biotin-labeled DNA pellet in 5 l of Cot-1 DNA (2 g/l) and transfer to a 200-l PCR tube. Overlay the solution with mineral oil, denature by incubation at 95 °C for 5 min, and incubate at 65 °C for 15 min. Add 5 l of 2 hybridization buffer and incubate further at 65 °C for 6 h.
 Critical step

Primary selection and capture of hybrids
10. Deliver 1 g of linkered genomic DNA from Step 6 (1 g/l) in 5 l of water into a 200-l PCR tube and overlay with mineral oil. Denature the DNA at 95 °C for 5 min and incubate at 65 °C for 15 min. Add 5 l of 2 hybridization buffer and transfer the entire sample to the tube containing the Cot-1−blocked BAC DNA from Step 9. Incubate the hybridization reaction at 65 °C for a further 70 h (C₀t_1/2 of 25).

11. In a 1.5-ml microcentrifuge tube, wash 6.7 10⁷ (100 l) of streptavidin-coated beads twice in 200 l Streptavidin bead binding buffer. After each wash, remove the beads from the binding buffer with a magnetic separator.

12. Resuspend the beads in 150 l Streptavidin bead binding buffer and add the hybridization buffer to the bead suspension. Carry out binding at 15−25 °C for 30 min with periodic gentle mixing to ensure the beads remain evenly suspended.

13. Remove the beads from the binding buffer using a magnetic separator and discard the supernatant. Wash the beads once, at 15−25 °C for 15 min, in 1 ml of 1 SSC with 0.1% SDS, and then three times, each at 65 °C for 15 min, in 1 ml 0.1 SSC with 0.1% SDS.

14. To elute the genomic DNA hybridized to the BAC, add 100 l of 0.1 M NaOH to the beads and incubate at 15−25 °C for 10 min. Remove the beads from the elution mixture using a magnetic separator.

15. Transfer the supernatant to a fresh 1.5-ml microcentrifuge tube containing 100 l of 1 M Tris-HCl (pH 7.5) and desalt the solution by spin-column chromatography through Sephadex G-50 resin.

Amplification of the primary selected DNA
16. Set up three separate amplification reactions in 200-l PCR tubes:

 Critical step

17. Amplify the three reactions according to the following program:

18. To determine which aliquot of template (1 l, 5 l or 10 l) yields the best range of amplified products, analyze the reaction products by agarose gel electrophoresis, including as control 1 l of the DNA sample set aside in Step 1.
It is advisable to transfer the DNA from the gel by Southern blotting and hybridize the membrane with a single-copy probe to assess enrichment of the template. The primary selection should yield approximately one thousand-fold enrichment.
 Critical step

19. Select the volume of template identified in Step 18 and use this volume to prepare a series of eight amplification reactions according to the conditions described in Step 16. Carry out the amplification as described in Step 17.

20. Purify the amplification products using a QIAquick PCR purification kit. Pool the eluted samples and determine the concentration of amplified primary selected DNA by spectrophotometry.

21. Remove a volume of DNA equivalent to 1 g and reduce this volume to 5 l in a speed vacuum concentrator. Set aside 1 l (at least 200 ng) of the primary selected material for comparison with the secondary selection products.

Further enrichment by secondary selection
22. Set up a reaction to biotinylate and block 100 ng of purified BAC DNA with Cot-1 DNA, following Steps 7−9.

23. Transfer the primary selected DNA (from Step 21) to a 200-l PCR tube and overlay with mineral oil. Denature the sample at 95 °C for 5 min and then incubate at 65 °C for 15 min. Add 5 l of 2 hybridization buffer and transfer the sample to the tube containing the Cot-1−blocked BAC (from Step 22). Incubate the hybridization reaction at 65 °C for 70 h.

24. After hybridization, bind the sample to streptavidin-coated magnetic beads and wash; recover the selected DNA as described for the primary selection (Steps 11−15).

Amplification of secondary selected material
25. Set up three amplification reactions as described in . Transfer 1 l of the secondary selected DNA (Step 24) into one tube, 5 l into the second and 10 l into the third. Add water to each tube to a final volume of 50 l and amplify the three reactions according to the program described in Step 17.
 Critical step

26. To determine which aliquot of template (1 l, 5 l or 10 l) yields the best range of amplified products, analyze the products by agarose gel electrophoresis. Include as controls 1 l of the starting genomic DNA (Step 1) and 100 ng of primary selected DNA (Step 21).
It is advisable to transfer the DNA from the gel by Southern blotting and hybridize the membrane with a single-copy probe to assess enrichment of the template. The secondary selection should yield an approximately tenfold further enrichment.
 Critical step

27. Select the volume of template identified in Step 26 and prepare a series of eight amplification reactions according to the conditions described in Step 16. Amplify the reactions according to the following program:

If possible, limit the number of PCR cycles to 20 or fewer to minimize the chances of PCR-induced sequence errors.
 Critical step

28. Purify the amplification products using a QIAquick PCR purification kit. Pool the eluted samples and determine the concentration of amplified secondary selected DNA by spectrophotometry.

EcoRI digestion and purification of the amplified products
29. Set up a reaction to digest the secondary selection material:

Incubate the reaction at 37 °C for 3 h.
The linker (annealed oligonucleotides 3 and 4) is designed with an internal EcoRI site to facilitate cloning of amplified products after selection.

30. Resolve the digested DNA by electrophoresis through a 1% TAE agarose gel. Isolate the selected material ranging from 300 base pairs (bp) to 2 kb by cutting out and removing the appropriate portion of the gel. Purify the sample from the gel using a QIAquick gel extraction kit and quantify the eluted material by spectrophotometry.

31. Transfer a volume of DNA (from Step 30) that corresponds to 100 ng into a 200-l PCR tube and reduce the volume to 3 l in a speed vacuum concentrator.

32. Clone the DNA fragments into the EcoRI site of pAMP1 and transform the recombinant plasmids into E. coli strain DH10B.
The transformants may now be analyzed by sequencing to identify the presence of sequence variations within the cloned region.

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TROUBLESHOOTING
Problem: The material fails to amplify.
[Step 2]
Solution: Test 100 ng of the genomic DNA containing linkers in a PCR, as described in Steps 16−17. As negative control, run a parallel PCR with 1 l digested DNA without linkers (from Step 1). The DNA with linkers should yield a smear of products on an agarose gel with an average size of 500 bp, whereas the DNA without linkers should not. If the DNA containing linkers fails to amplify, it is likely that there has been a failure of the fill-in reaction (Step 2) or of the ligation (Step 5). Repurify the genomic DNA and start from the beginning of the protocol.

Problem: The material fails to amplify.
[Step 5]
Solution: Test 100 ng of the genomic DNA containing linkers in a PCR, as described in Steps 16−17. As negative control, run a parallel PCR with 1 l digested DNA without linkers (from Step 1). The DNA with linkers should yield a smear of products on an agarose gel with an average size of 500 bp, whereas the DNA without linkers should not. If the DNA containing linkers fails to amplify, it is likely that there has been a failure of the fill-in reaction (Step 2) or of the ligation (Step 5). Repurify the genomic DNA and start from the beginning of the protocol.

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CRITICAL STEPS
Preparation of the genomic DNA and double-stranded linkers, Step 1 Alternatively, random shearing, size selection and repair of genomic DNAs may easily be incorporated into this protocol for regions that lack a good distribution of restriction enzyme sites.

Biotinylation and blocking of the BAC DNA, Step 7 Check that the BAC DNA has been efficiently biotinylated by removing a small aliquot (1% of the total) and testing its binding to streptavidin beads. Monitor the radioactive tracer on the beads and in the supernatant with a Geiger counter or by Cherenkov counting. The ratio of bound to unbound counts should be at least 8:1. Do not proceed if this ratio is not achieved. If the ratio is lower, either repurify the BAC DNA by extraction with phenol/chloroform and purification on Sephadex spin columns, or increase the ratio of biotin-16-dUTP to unlabeled nucleotide in the labeling reaction (Step 7). However, there are upper limits to this latter solution, as too many biotin moieties bound to the BAC DNA can sterically hinder hybridization (Step 9).

Biotinylation and blocking of the BAC DNA, Step 9 Check that the BAC DNA has been efficiently biotinylated by removing a small aliquot (1% of the total) and testing its binding to streptavidin beads. Monitor the radioactive tracer on the beads and in the supernatant with a Geiger counter or by Cherenkov counting. The ratio of bound to unbound counts should be at least 8:1. Do not proceed if this ratio is not achieved. If the ratio is lower, either repurify the BAC DNA by extraction with phenol/chloroform and purification on Sephadex spin columns, or increase the ratio of biotin-16-dUTP to unlabeled nucleotide in the labeling reaction (Step 7). However, there are upper limits to this latter solution, as too many biotin moieties bound to the BAC DNA can sterically hinder hybridization (Step 9).

Amplification of the primary selected DNA, Step 16 It is very important to use the highest-quality and highest-fidelity thermostable polymerase. It is also advisable, if possible, to limit the number of amplification cycles, to reduce the possibility that resulting DNA sequences may be riddled with PCR-induced sequence errors rather than true genomic DNA sequence variants.

Amplification of the primary selected DNA, Step 18 At this point one may either qualitatively or quantitatively assess the degree of enrichment (or one may proceed with blind faith). To gain a qualitative estimate, hybridize the Southern blot of genomic DNA and primary selected material with a radiolabeled single-copy DNA fragment derived from within the genomic region carried in the BAC (for example, a PCR fragment of a few hundred base pairs). The resulting autoradiograph should show a very high degree of enrichment in the primary selected material (1,000-fold). If this is not the case, do not proceed with the secondary selection; start from the beginning of the protocol and double check DNA purity, concentrations, and bead binding parameters. A quantitative assessment of enrichment can be achieved by deriving a few thousand molecular clones from the primary selected material (Step 21) and conducting colony lift hybridizations with the single copy probe.

Amplification of secondary selected material, Step 25 It is very important to use the highest-quality and highest-fidelity thermostable polymerase. It is also advisable, if possible, to limit the number of amplification cycles, to reduce the possibility that resulting DNA sequences may be riddled with PCR-induced sequence errors rather than true genomic DNA sequence variants.

Amplification of secondary selected material, Step 26 At this point one may either qualitatively or quantitatively assess the degree of enrichment (or one may proceed with blind faith). To gain a qualitative estimate, hybridize the Southern blot of genomic DNA and primary selected material with a radiolabeled single-copy DNA fragment derived from within the genomic region carried in the BAC (for example, a PCR fragment of a few hundred base pairs). The resulting autoradiograph should show a very high degree of enrichment in the primary selected material (1,000-fold). If this is not the case, do not proceed with the secondary selection; start from the beginning of the protocol and double check DNA purity, concentrations, and bead binding parameters. A quantitative assessment of enrichment can be achieved by deriving a few thousand molecular clones from the primary selected material (Step 21) and conducting colony lift hybridizations with the single copy probe.

Amplification of secondary selected material, Step 27 It is very important to use the highest-quality and highest-fidelity thermostable polymerase. It is also advisable, if possible, to limit the number of amplification cycles, to reduce the possibility that resulting DNA sequences may be riddled with PCR-induced sequence errors rather than true genomic DNA sequence variants.

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Natureproducts is an online service detailing information about specific products used in this article, you can view the product descriptions, request information and compare with other similar products. The products used are listed in alphabetical order. A-Z product listing [-32P]dCTP (Amersham) Biotin-16-dUTP (Enzo Biochemicals) Cot-1 DNA (Invitrogen) DH10B electrocompetent Escherichia coli (Invitrogen) DNA polymerase/DNase I (Roche) dNTP solution (Invitrogen) See more natureproducts

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