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Fosmid Libraries for Genomic Structural Variation Detection

William F. Donahue¹, Heather M. Ebling¹

¹Agencourt Bioscience, Beverly, Massachusetts

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 5.20

DOI:

10.1002/0471142905.hg0520s54

Online Posting Date:

July, 2007

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Abstract

Fosmid libraries have demonstrated their utility for a number of applications. These include filling gaps between BACs and small insert libraries in sequence assemblies, performing hybridization/screening studies to isolate functional elements within the genome (Vergin et al., 1998), and detecting insertions, deletions, and rearrangements in structural variation studies (Tuzun et al., 2005). This unit covers the basic methodologies for the construction of fosmid libraries with tight insert sizes suitable for these applications. Basic Protocol 1 covers the shearing, size selection, and recovery of DNA from a pulsed-field gel. Basic Protocol 2 covers the cloning of insert DNA into the fosmid vector, packaging of DNA into infective phage particles, and the infection/transformation of bacteria. A commentary section is provided, which outlines many of the critical parameters involved in fosmid library construction, along with some additional background information and a section discussing anticipated results. Curr. Protoc. Hum. Genet. 54:5.20.1-5.20.18. © 2007 by John Wiley & Sons, Inc.

Keywords: fosmid cloning; fosmid library; fosmid; fosmid protocol; structural variation detection

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Unit Introduction
Basic Protocol 1: Preparing DNA for Ligation into pCC2FOS Vector
Basic Protocol 2: Vector Ligation, DNA Packaging, and Cell Transformation
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Preparing DNA for Ligation into pCC2FOS Vector

Materials

Genomic DNA
3 M sodium acetate, pH 5.5 (Sigma)
70% and 100% ethanol (Sigma)
GlycoBlue (Ambion)
End-It DNA End-Repair kit (Epicentre) containing:
- End-Repair enzyme mix: T4 DNA polymerase and T4 polynucleotide kinase
- 10× End-Repair buffer
- dNTP solution
- ATP
LoTE buffer, pH 7.5 and 8 (see recipe)
Phenol/chloroform (Sigma; also see appendix 3C)
Chloroform/isoamyl alcohol (Sigma; also see appendix 3C)
10× TBE buffer (appendix 2D)
Agarose (Seakem Gold, Cambrex or equivalent)
10× BlueJuice gel loading buffer (Invitrogen)
Monocut DNA marker (New England Biolabs)
1-kb extension ladder (Invitrogen)
SYBR Gold gel dye (Invitrogen)
1× TAE buffer (appendix 2D)

HydroShear and large shearing assembly (Genomic Solutions)
1.5- and 1.7-ml microcentrifuge tubes
Refrigerated centrifuge (Eppendorf or equivalent)
PCR strip tubes
Thermal cycler
CHEF Mapper XA pulsed-field electrophoresis system (BioRad)
10-well comb (4-cm wide, 1.5-mm thick, adjustable height)
Razor blades (VWR)
Platform shaker, 37°C
Gel reader (e.g., DarkReader)
Dialysis membrane tubing (Spectra-Por 15,000 MWCO, VWR)
10-ml serological pipet
Weighted tubing closures (VWR)
Gel box (e.g., Owl model B3)
Microcon spin columns (YM-100, Millipore)
UV spectrophotometer
Additional reagents and equipment for extraction and precipitation of DNA (appendix 3C)

Basic Protocol 2: Vector Ligation, DNA Packaging, and Cell Transformation

Materials

CopyControl HTP Fosmid library production kit (CCFOS059, Epicentre) containing:
- CopyControl pCC2FOS vector
- End-Repair enzyme mix
- End-Repair 10× buffer
- dNTP mix
- Fast-Link DNA ligase
- Fast-Link 10× ligation buffer
- ATP solution
- GELase gel-digesting preparation
- GELase 50× reaction buffer
- MaxPlax lambda packaging extracts
- Control DNA
- Ligated lambda control DNA
- EPI300 plating strain
- Control lambda plating strain
- CopyControl induction solution
T4 DNA ligase (Invitrogen)
10× T4 DNA ligation buffer (see recipe)
Phage dilution buffer (see recipe)
Chloroform (Sigma)
LB medium (appendix 2D) with 10 mM MgSO₄ (appendix 2D)
10 mM MgSO₄

16°, 30°, and 70°C water bath
Microcon YM-100 column (100 k MWCO; Millipore)
15- and 50-ml centrifuge tubes (VWR)
37°C shaker
2-ml cryotubes (Nunc)

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Figures

Figure 5.20.1

Diagram of the pCC2FOS vector, adapted from EPICENTER Product Literature (2006) with permission from EPICENTER Technologies Corporation. All rights reserved. The pCCFOS2 vector allows for blunt-end cloning of DNA for fosmid libraries. The vector contains a single cos site and an E. coli. F-factor-derived single-copy origin of replication as well as an inducible oriV replication origin. FP and RP sequencing primer binding sites are located immediately adjacent to the vector/insert junction to minimize the inclusion of wasteful vector sequence.

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Figure 5.20.2

Excision of marker containing lanes. Insert DNA can be size selected without exposure to SYBR gold or ethidium bromide by removing the marker containing regions from the main body of the gel and staining them separately. The gel can then be reassembled and band excised on a DarkReader. Alternatively, the two maker regions can be placed together on the illumination source and marked (cut) at the points at which bands will be excised. The gel can then be reassembled and cut without an additional light source.

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Figure 5.20.3

Pulsed-field gel post-excision. Marker containing regions were removed from this 0.8% pulsed-field gel and stained to allow for sizing of insert DNA without exposure to UV light or SYBR gold. Horizontal lines show the approximate regions excised to produce fosmid libraries (three slices). It is important to excise bands straight across length of gel to minimize insert size variability. After removing the areas of interest from the main body of the gel it can and stained, reassembled with the marker regions, and photographed as shown here.

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Figure 5.20.4

HydroShear DNA fragmentation. DNA test shear visualized on a 0.8% pulsed-field agarose gel. Genomic DNA was sheared in a 125-µl volume for 20 cycles using a Genomics Solutions HydroShear. The size of the sheared fragment increases as the speedcode is increased. When shearing with the “large” HydroShear assembly, the size of the sheared DNA is also determined by the concentration of the DNA sample in addition to the speed code setting. Lane 4 is a shear with 2 µg of DNA (16 ng/µl). Lane 8 contains DNA sheared with the same volume and speed code but at a concentration of 160 ng/µl.

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Figure 5.20.5

Fosmid insert size variability. These histograms plot the size of the insert versus the number of reads per insert of a given size. Insert sizes were calculated by mapping paired-end fosmid reads against the human genome. The low, middle, and upper slice histograms were derived from a sample of sequenced clones from libraries produced from the three gel slices taken from the gel in Figure 5.20.3.

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Literature Cited

Literature Cited
	Collins, J. and Hohn, B. 1978. Cosmids: A type of plasmid gene-cloning vector that is packageable in vitro in bacteriophage heads. Proc. Natl. Acad. Sci. U.S.A. 75:4242-4246.
	EPICENTER Product Literature (2006). EPICENTER Product Literature #171: CopyControl TM Fosmid Library Production Kit, 2006, EPICENTER Technologies Corporation. All rights reserved.
	Feiss, M., Kobayashi, I., and Widner, W. 1983. Separate sites for binding and nicking of bacteriophage DNA by terminase. Proc. Natl. Acad. Sci. U.S.A. 80:955-959.
	Hohn, B. and Murray, K. 1977. Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proc. Natl. Acad. Sci. U.S.A. 74:3259-3263.
	Jones, P.G., VanBogelen, R.A., and Neidhardt, F.C. 1987. Induction of proteins in response to low temperature in Escherichia coli. J. Bacteriol. 169:2092-2095.
	Kandror, O. and Goldberg, A.L. 1997. Trigger factor is induced upon cold shock and enhances viability of Escherichia coli at low temperatures. Proc. Natl. Acad. Sci. U.S.A. 94:4978-4981.
	Kandror, O., Deleon, A., and Goldberg, A.L. 2002. Trehelose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures. Proc. Natl. Acad. Sci. U.S.A. 99:9727-9732.
	Kim, U., Shizuya, H., deJong, P.J., Birren, B., and Simon, M.I. 1992. Stable propagation of cosmid sized human DNA inserts in an F–factor-based vector. Nucl. Acids Res. 20:1083-1085.
	Phadtare, S. and Inouye, M. 2004. Genome-wide transcriptional analysis of the cold shock response in wild-type and cold-sensitive, quadruple-csp-deletion strains of Escherichia coli. J. Bacteriol. 186:7007-7014.
	Shizuya, H., Birren, B., Kim, U., Mancino, V., Slepak, T., Tachiiri, Y., and Simon, M. 1992. Cloning and maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. U.S.A. 89:8794-8797.
	Stenberg, N., Tiemeir, D., Enquist, L. 1977. In vitro packaging of a lambda Dam vector containing EcoRI fragments of Escherichia coli and phage P1. Gene. 1:255-280.
	Thomas, M., Cameron, J.R., and Davis, R.W. 1974. Viable molecular hybrid of bacteriophage lambda and eukaryotic DNA. Proc. Natl. Acad. Sci. U.S.A. 71:4579-4583.
	Tuzun, E., Sharp, A.J., Bailey, J.A., Kaul, R., Morrison, V.A., Pertz, L.M., Haugen, E., Hayden, H., Albertson, D., Pinkel, D., Olson, M.V., and Eichler, E.E. 2005. Fine scale structural variation of the human genome. Nat. Genet. 37:727-732.
	Weiczorek, D.J. and Feiss, M. 2001. Defining cosQ, the site requirement for termination of bacteriophage DNA packaging. Genetics 158:495-506.
	Wild, J., Hradenca, Z., and Szybalski, W. 2002. Switching from single-copy to high-copy vectors and genomic clones. Genome Res. 12:1434-1444.
	Vergin, K.L., Urbach, E., Stein, J.L., DeLong, E.F., Lanoil, B.D., and Giovannoni, S.J. 1998. Screening of a fosmid library of marine environmental genomic DNA fragments reveals four clones related to members of the order Planctomycetales. Appl. Environ. Microbiol. 64:3075-3078.