The construction of detailed gene-linkage maps of complex genomes facilitates the localization of genes to specific regions on chromosomes. Further, precise loclalization of specific genes requires cloning of high-molecular-weight DNA if it is to be done efficiently. Two methods commonly used are to either insert large genomic DNA fragments into cosmid vectors, which are then packaged into Phage lambda heads and propagated in Escherichia coli,1 or into yeast artificial chromosome (YAC) vectors propagated in Saccharomyces cerevisiae.2 The use of cosmids results in high transformation efficiencies, but insert size is limited by the size of the Phage lambda head to 45-48 kilibase pairs (kbp). The advantage of using YAC vectors is their ability to accommodate DNA as large as 200-800 kbp. Disadvantages are the low transformation efficiency, which decreases with insert size; the need to process transformants individually prior to screening; and the difficulty in obtaining large amounts of recombinant DNA from transformed cells.3
To overcome some of the problems associated with using cosmid or YAC systems, a novel method for cloning and packaging DNA fragments using a Bacteriophage P1 system has been developed3 that offers the ability to clone large genomic DNA fragments of between 70-95 kb in size with efficiencies approaching those of cosmids. In addition, the P1 DNA Packaging System uses host E. coli strains and in vitro packaging extracts obtained from strains that are deficient in restricition and recombination abilities. These prevent the degradation and recombination of methylated genomic DNA.4
Principle of the Method
Using a strategy analogous to Phage lambda packaging, partially digested and size selected genomic DNA between 70 and 95 kb is ligated onto linearized plasmid vector arms. The SacBII vector used contains a Phage P1 pac-cleavage site and two Phage P1 loxP recombination sites in addition to replication origins and an antibiotic resistance gene (Fig. 1) The recombinant vector is cleaved at the pac-cleavage site in a "Pacase" extract and the resulting DNA is then inserted into an empty P1 Phage head using a second extract containing phage packaging proteins. The attachment of P1 phage tails to the heads results in the formation of infectious recombinant phage particles that are then used to infect a restriction minus Escherichia coli host strain containing an expressed cre gene.
After injection into the host strain, the recombinant DNA is circularized between the two Phage P1 loxP sites by Cre recombinase. DNA which does not circularize is degraded by host nucleases. The circular DNA molecule now replicates and is maintained stably at one copy per host cell by the P1 Plasmid replicon.
Prior to alkaline lysis plasmid isolation,5 the recombinant plasmid
copy number is increased more than 25-fold by isopropyl B-Dthiogalactopyranoside
(IPTG) induction of the lac promoter
controlled high-copy P1 lytic replicon. A schematic detailing the
P1 DNA packaging strategy is shown in Figure 2.
The SacBII vector is digested with restriction endonucleases Scal
and BamHI and the ends dephosphorylated. This generates two vector
"arms," one consisting of the "short" Scal to BamHI fragment and
the other the "long" Scal to BamHI fragment. Genomic DNA is
partially digested with BamHI or other compatible end restriction
endonucleases and size-selected on a sucrose gradient. Fragments
between 70 kb and 95 kb in length are isolated and ligated to the
vector arms, generating a series of linear molecules. If ligation
occurs between two "short" arms, the resulting molecule will
neither contain the origins of replication nor the kanR gene, and
will be nonviable. If both arms are "long," there will be no pac
site, and no packaging into the phage heads will occur. The only
viable recombinant will be one consisting of the insert sequence
flanked by both a short and long arm.
Phage P1 uses a headful packaging strategy and can accommodate a
total DNA length of approximately 110-115 kb. Any inserts longer
than 95-100 kb will result in truncation of the packaged DNA before
the distal loxP site is inserted, and the molecule will be unable
to circularize upon injection into the host. Once injected into the
cre+ host cell, the cre protein circularizes the injected DNA at
the loxP sites, and DNA now replicates using the plasmid origin of
replication. Propagation of cells on sucrose containing media only
permits growth of colonies with genomic DNA inserts (positive
selection). Plasmid copy number is increased by induction with
IPTG. The recombinant DNAs are then isolated as plasmids using
traditional methods.
Phage P1 uses a headful packaging strategy. Once the phage head is filled with DNA (about 110-115 kb), a "headful cut" occurs, cleaving any remaining DNA away from the head before it is packaged. This packaging mode suggests that if the insert DNA is too large (>95 kb), it will be packaged but not recovered in bacteria because the "headful" packaging process will terminate before the distal loxP is incorporated into the phage head. It also suggests that DNA too small to generate a headful when inserted into the vector should not be packaged into phage particles.
However, it has been observed that DNA less than a P1 "headful" is packaged in vitro in viable phage particles with an efficiency of about 10-15 percent that of "headful" DNA (Pierce & Sternberg, manuscript in preparation). While it is not completely clear why this should occur, it points out the need to size-select the insert DNA on sucrose gradient before attempting to ligate it to vector in order to maximize the recovery of large inserts. In particular, size fractions that maximize DNA fragments in the 70-95 kb range, and minimize the presence of shorter fragments, should be used. The presence of small fragments is undesirable also because it increases the possibility that they will be ligated together and then recovered in one clone. This would significantly complicate subsequent screening and gene localization processes.
Finally, as the second stage packaging extract contains about 5-10 percent small heads (headful size 47 kb), small DNA fragments can be recovered by a headful packaging process in phage particles containing these heads.
An 11 kb "stuffer" region of Adenovirus type 2 DNA has been engineered into the pAd10-SacBII cloning vector. It is designed to provide a segment of DNA in which the "headful" cut can be made. Using the P1 DNA Packaging System, genomic DNA from 70-95 kb can be readily cloned and manipulated. The major advantages of the P1 DNA packaging method over other genomic cloning methods are: 1) the ability to clone inserts two to three times the size of those used with cosmids and lambda vectors, 2) no rearrangement or deletion of methylated DNA occurs because of the use of restriction-minus host strains, and 3) recombinant DNA is easily recovered as plasmids for further screening and minipulations.
P1 Cloning Vectors
One of the key components in the construction of a P1 DNA library is the genetically engineered P1 plasmid vector. The original vector was pNS582tet14Ad10 (see Fig. 3). A new improved, more versatile version of the Ad10 vector has been constructed. This vector is designated pAd10-SacBII (see Table 1) and will be available soon.
REFERENCES:
Du PONT'S P1 DNA Packaging System
ORDERING INFORMATION
Call 1-800-551-2121 1,1 at the prompt
NEP-113
NEP-113 contains all the components found in both NEP-113C and NEP- 113P, listed below. (NOTE: NEP-113 does not include "SacBII" positive selection cloning vector.)
NEP-113C
The P1 DNA Cloning System contains the components needed for ligating size-fractionated genomic DNA into the P1 "SacBII" cloning vector (ordered as NEP-113V). The kit includes: P1 control "Ad-10" vector, T4 DNA ligase and ligation buffer, Calf Intestinal Alkaline Phosphatase, and high molecular weight DNA markers (used for selecting DNA of optimal length for cloning and packaging).
NEP-113P
The P1 DNA Packaging System consists of all components needed for five packaging reactions of genomic DNA to P1 vector clones, including the phage P1 Head/Tail extract, the Pacase extract for cleaving the recombinant DNA and guiding it into phage heads, the recombination and restriction-minus bacterial plating host strains, and all necessary buffers.
NEP-113V
New P1 cloning vector pNS582tet14-Ad10-SacBII, the positive selection cloning vector used for genomic DNA library generation. Must be ordered separately; not included with NEP-113 or NEP-113C. Available soon.