ABSTRACT
We have developed a novel method to clone and sequence minute quantities of DNA.
The method was applied to sequence a 180 kb plasmid pNL1. The first step was
the production of a size distributed population of DNA molecules that were
derived from the 180 kb plasmid pNL1. The first step was accomplished by a
random synthesis reaction using Klenow fragment and random hexamers tagged with a T7
primer at the primer 5
'
-end (T7-dN
6
, 5
'
-GTAATACGACTCACTATAGGGCNNN- NNN-3
'
). In the second step, Klenow-synthesized molecules were amplified by PCR using T7 primer (5
'
-GTAATACGACTCACTATAGGGC-3
'
). With a hundred nanograms starting plasmid DNA from pNL1, we were able to generate Klenow-synthesized molecules with sizes ranging from 28 bp to >23 kb which were
detectable on an agarose gel. The Klenow-synthesized molecules were then used as templates for standard PCR with T7
primer. PCR products of sizes ranging from 0.3 to 1.3 kb were obtained for
cloning and sequencing. From the same Klenow-synthesized molecules, we were also able to generate PCR products with
sizes up to 23 kb by long range PCR. A total 232.5 kb sequences were obtained
from 593 plasmid clones and over twenty putative genes were identified.
Sequences from these 593 clones were assembled into 62 contigs and 99
individual sequence fragments with a total unique sequence of 86.3 kb.
Sphingomonas
F199 was isolated from sediments at a depth of 407 m (
1
). This bacterium has the ability to use toluene, all isomers of xylene,
p
-cresol, naphthalene, salicylate and benzoate as sole carbon and energy
sources (
1
). It harbors two megaplasmids of 180 kb (pNL1) and 475 kb (pNL2) (
2
). An initial catabolic screening study with a cosmid library generated from
pNL1 has located catechol 2,3-dioxygenase activity on pNL1 (
2
). To explore the potential of using pNL1 for subsurface bioremediation, we
initiated a project to sequence pNL1 completely.
Partial restriction digestion or mechanical shearing of DNA templates to obtain
DNA fragments with sizes ~1 kb are the currently-preferred methods for the construction of plasmid libraries for
sequencing. However, such methods often require a large quantity (50-100 [mu]g) of starting DNA material and are labor intensive. Two methods
which are the whole genome PCR (
3
) and primer-extension preamplification (PEP) (
4
) have been developed to amplify the whole genome DNA from a small quantity of DNA material.
Amplified materials were used for the isolation of specific DNA sequences and
for genetic analysis (
3
,
4
). The whole genome PCR method still requires restriction digestion or
sonication to generate small fragments, which are then ligated to a linker for
PCR amplification. The PEP approach uses a mixture of 15-base oligonucleotides as primers for PCR amplification. However, the
efficiency of the amplification is very low and PEP approach does not amplify
enough material to make a plasmid library that is adequate for sequencing.
To increase the amplification efficiency of PEP, a tagged random primers PCR (T-PCR) method (
5
) was developed to amplify efficiently from small quantities of DNA samples with
sizes ranging from 400 bp to 1.6 kb. This method involves two PCR reactions
with tagged random primers containing nine to 15 random bases at the 3'-end and a constant 17 bp at the 5'-end. In the first PCR step, the tagged random primer
is used to generate products with tagged primer sequences at both ends, which
are achieved by using a low annealing temperature in the PCR cycles. Excess
tagged primers are then removed. In the second PCR step, the primer with the
constant 17 bp sequence is used to amplify PCR products from the first PCR
step. Since tagged primers with 12 or more random bases will generate non-specific products, presumably resulting from primer-primer extensions or less efficient elimination of these longer
primers during the filtration step (
5
), it would be advantageous to use a tagged random primer with shorter random
bases. In this report, random synthesis was achieved by using Klenow fragment
and random hexamer tagged with T7 primer at the primer 5'-end. Klenow-synthesized molecules were then amplified with T7 primer. As
far as we know, this is the first report of the cloning and sequencing of
randomly amplified PCR products to assess its coverage of the original
template.
pNL1 is a 180 kb plasmid isolated from a subsurface bacterium
Sphingomonas
F199, which can utilize a variety of aromatic compounds as sole carbon sources
(
6
). Preparation of the plasmid pNL1 was described previously (
2
). T7 primer (5'-GTAATACGACTCACTATAGGGC-3') and tagged random hexamer, T7 primer-dN6 (5'-GTAATACGACTCACTATAGGGCNNNNN-3') were synthesized using
an Applied Biosystems RNA/DNA synthesizer 392 (Perkin Elmer, Foster City, CA).
TRHA products were blunt-end ligated into the pCRscript cloning vector (Stratagene, La Jolla, CA)
using standard cloning procedures (
7
). Ligated products were used to transform XL1-Blue supercompetent cells (Stratagene, La Jolla, CA) and transformed cells
were then plated onto LB agar plates containing 200 [mu]g/ml ampicillin, X-gal and IPTG (X-gal plates). White colonies which appeared on X-gal plates were picked individually with pipette tips into a 96-well microtiter plates containing 2YT medium (16 g/l
tryptone, 10 g/l yeast extract and 5 g/l NaCl). The cells were allowed to grow
in the microtiter plates at 37oC without shaking for 4 h. A 2 [mu]l cell culture was then directly transferred to a 50 [mu]l PCR reaction mix which contained 1.5 mM MgCl
2
, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.2 mM dNTP, 0.2 [mu]M RSP (5'-GGAAACAGCTATGACCATGA-3') and SP (5'-GTAAAACGACGGCCAGT-3') primers, and
1.25 U ampliTaq (LD) (Perkin Elmer, Foster City, CA). The PCR reactions were first heated to 72oC for 10 min, then 30 cycles of the following temperature profile: 95oC for 1 min, 60oC for 1 min and 72oC for 2 min. PCR products were analyzed on a 1.2% agarose
gel.
Cosmid clones of pNL1 were digested with
Eco
RI and the resulting fragments were separated by electrophoresis on a 0.8%
agarose gel. The gel was then blotted onto Duralon membrane and UV cross-linked. Fluoresceinated DNA probes were prepared with Prime-It Fluor Fluorescence Labelling Kit (Stratagene, La Jolla, CA)
according to manufacturer's instruction. Hybridization was done at 60oC in 15 ml QuickHyb solution (Stratagene, La Jolla, CA) for 3 h. The
hybridization signal was detected with Illuminator Non-radioactive Detection System (Stratagene, La Jolla, CA). Fluorescent
substrate ATTOPHOS (JBL Scientific INC., San Luis Obispo, CA) was used in place
of CSPD chemiluminescent substrate and hybridization signals were detected with
FluorImager SI (Molecular Dynamics, Sunnyvale, CA).
Klenow-synthesized molecules were used as template for long range PCR using
TaqPlus DNA polymerase (Stratagene, La Jolla, CA). Two different reactions were
set up, one in a 100 [mu]l low salt buffer (20 mM Tris-HCl, pH 8.75, 10 mM KCl, 10 mM (NH
4
)SO
4
, 2 mM MgSO
4
), the other in high salt buffer (20 mM Tris-HCl, pH 9.2, 60 mM KCl, 2 mM MgCl
2
), which in addition, contained 0.2 mM dNTP and 0.5 [mu]M T7 primer. The PCR reactions were heated to 72oC for 10 min and 5 U TaqPlus DNA polymerase was then added. The
reactions were then subjected to 30 cycles of PCR reaction with the following
temperature profile: 94oC for 30 s, 60oC for 30 s and 72oC for 15 min.
Reaction products after the Klenow (Klenow-synthesized molecules) and PCR (TRHA products) steps were analyzed by
agarose gel electrophoresis (Fig.
2
). A 10 [mu]l volume of Klenow-synthesized molecules from pNL1 were separated on a 1% agarose gel
(Fig.
2
, lanes 1 and 2). The DNA quantities were amplified during the Klenow random
synthesis step. Klenow-synthesized molecules appeared as a smear with sizes ranging from that of
the primer to >23 kb. The amount of products were significantly increased by two rounds of random synthesis with Klenow fragment.
Klenow-synthesized molecules with or without excess T7-dN
6
removed were then used as templates for subsequent PCR amplification. The
relative size and yield of TRHA products increased when the excess T7-dN
6
primer was removed (Fig.
2
, lanes 5 and 6). This is probably due to primer dimer formation during PCR
amplification. Similarly, we also found that the yield of Klenow-synthesized molecules decreased when the concentration of T7-dN
6
primer increased from 0.9 to 2.7 [mu]M (data not shown).
We were able to obtain PCR products in the range from 0.3 to 1.3 kb from Klenow-synthesized molecules derived from either one or two cycles of Klenow
random synthesis (Fig.
2
, lanes 3-6). This result implies that two possible events might have taken place
during the one cycle of Klenow random synthesis. Firstly, Klenow fragment is
able to use the first synthesized DNA strand as template to generate a second
strand DNA without requiring a second denaturing step. Secondly, the template, being denatured, provides
sites for first-strand annealing in both directions and products with 3'-ends complementary to each other will be present. In the
former case, the second strand DNA will have two T7 primer sites in opposite
orientation at both ends for PCR reaction. In the second case, Klenow-synthesized molecules with 3' complementary to each other will be extended in the PCR step. As a
result, products with T7 sequences at both ends will be available for
subsequent PCR amplification. Furthermore, the average size of PCR products
using template from one cycle of Klenow random synthesis (Fig.
2
, lane 5) was comparatively larger than that from two cycles of Klenow random
synthesis (Fig.
2
, lane 6). This size difference indicated that two cycles of Klenow random
synthesis may have biased towards smaller fragments because smaller molecules
with T7 primer sites at both ends are preferentially-synthesized.
The coverage of the TRHA products was tested using a miniset of cosmid clones
from pNL1 plasmid (
1
). Six cosmid clones with an average insert size of 40 kb that provide
overlapping coverage of the 180 kb plasmid were completely digested with
Eco
RI and run on a 0.8% agarose gel (Fig.
3
A). The gel was Southern blotted and hybridized with fluoresceinated DNA probes
generated from the TRHA products (Fig.
2
, lane 5). Hybridization results (Fig.
3
B) showed that all the
Eco
RI fragments including those of ~500 bp were represented, but some fragments had low hybridization signals.
We speculate that those fragments with low intensity may represent the region
of the plasmid with non-random sequences such as repetitive sequences, GC or AT rich regions.
TRHA products (Fig.
2
, lane 5) were blunt-end ligated into pCRscript plasmid vector. The insert sizes of putative
clones were determined by direct PCR amplification using bacterial cells
carrying the recombinant plasmid as described in the Methods section. It was
found that the insert sizes of the clones range from 0.3 to 1.3 kb with an
average insert size of 0.5 kb (Fig.
4
). To further confirm that the insert is derived from pNL1, PCR amplified
products as shown in Figure
4
were dot-blotted onto a Nylon membrane and hybridized with a fluoresceinated DNA
probe generated from pNL1 plasmid DNA. Only clones without an insert as
revealed by PCR amplification did not have a hybridization signal (Fig.
5
). Thus, the results indicated that all the inserts were derived from pNL1.
A total
593 clones were sequenced with T3 primers using reagents from ABI PRISM Dye
terminator cycle sequencing ready reaction kit with ampliTaq DNA polymerase FS
and analyzed with an ABI377 sequencer (Perkin Elmer, Foster City, CA). After
trimming away the vector sequences, we obtained a total sequence of 232.5 kb
and over twenty putative genes were identified by searching non-redundant protein and nucleic acid databases at the National Center for
Biotechnology Information using Blastn and Blastx via the Internet. Using
sequence analysis software AssemblyLIGN (Oxford Molecular, Campbell ,CA),
sequences from these 593 clones were assembled into 62 contigs and 99
individual sequence fragments with a total unique sequence of 86.3 kb. Among
these 593 clones, 17 clones were concatemers which were resulted from the
annealing of T7 primer sites at the ends of two or more smaller Klenow-synthesized molecules. However, we did not detect any clones that align to
more than one contig.
To analyze the randomness of the TRHA product, the following statistical
analysis was performed. Assuming every fragment of the 180 kb pNL1 plasmid was
equally amplified, we can apply the Poisson distribution equation to calculate
the probability that a base is not sequenced. That is
P
= e
-
m
, where
m
is the sequence coverage (total sequence information obtained/size of the
original template) (
8
). In our case,
m
is equal to 1.29 ( i.e. 232.5/180). Thus, the expected
P
value will be 0.28, which means 28% of the pNL1 plasmid will not be sequenced.
However, the experimental result for the
P
value is 0.52 (i.e. 1-86.3/180) since we have obtained 86.3 kb unique sequence out of the 180
kb pNL1 plasmid. This discrepancy shows that the TRHA products are not fully
random. The hybridization result (Fig.
3
) also indicated that some regions of the plasmid pNL1 were under-represented in the TRHA products. To calculate the fraction (
f
) of pNL1 preferentially amplified as the TRHA products, we applied the
following formula,
P
= e
-
m
+ (1 -
f
). When
m
approaches infinity,
P
will equal (1 -
f
), which is the fraction of sequence under-represented in the TRHA products. By substituting the numbers from the
experimental result (
P
= 0.52 and
m
= 1.29),
f
is 0.76, which suggests that 76% of the total pNL1 plasmid sequence were
preferentially amplified and represented in the TRHA products. In contrast, a
mathematical model (
9
) suggested that only 37% of the DNA template are preferentially amplified in T-PCR (
5
). The difference is probably due to the Klenow step used in this protocol which
generates longer primary products for subsequent PCR amplification. The sizes
of the Klenow-synthesized molecules range from ~28 bp to >23 kb (Fig.
2
, lane 2). Using a long range PCR amplification protocol, we were able to
amplify PCR products up to 23 kb from the Klenow-amplified products (Fig.
6
). Such longer products are likely to cover regions of pNL1 not represented in
the TRHA products and will be useful for making a lambda library for gap
closure. Currently we are designing primers from the ends of each contigs and
trying to get the rest of the sequence of pNL1 from cosmid clones by primer
walkings. Alternatively, we are also trying to design tagged random hexamer
with a bias towards GC or AT so as to enrich regions which were under-represented in the TRHA products in this study.
A simple, fast and efficient method was described for making a plasmid library
from 100 ng of starting material. After sequencing 593 clones and obtaining
232.5 kb total sequences, we estimated that 76% of the original DNA template
were preferentially amplified and represented in the TRHA products.
This method can be applied to any situation where the amount of RNA or DNA is
limited. For example, only a small quantity of nucleic acid materials can be
obtained from (i) soil and subsurface environment with low biomass, (ii)
restriction DNA fragments purified from agarose gels or YAC chromosomal DNA
purified from pulsed-field gel electrophoresis, or (iii) a single cell. Large sequencing of BAC
(bacterial artificial chromosome), PAC (P1 clones) and cosmids are taking place
as the human genome project is going into its sequencing phase. Nebulization or
sonication are currently used to make plasmid libraries from cosmid clones, PAC
clones or BAC clones for sequencing. TRHA may be a timely method for making
plasmid subclone libraries for initial random sequencing. Since only a small
quantity of DNA is needed, miniprep of BAC, PAC or cosmid will be sufficient
for library construction. After initial sequencing, the rest of the sequence
may be obtained by PCR amplification of sequence gaps and primer walkings.
We thank Susan Varnum, Sarah Thruston, Toyoko Tsukuda and Rita Cheng for
discussions and reading the manuscript. We also thank Ellen Sisk for making the
tagged random hexamer. This work was supported by DOE contract DE-AC06-76RLO 1830.
A. Random synthesis with Klenow fragment and tagged random hexamer
. Two 100 [mu]l reactions were prepared, using 0.5* universal buffer (50 mM KOAc, 12.5 mM Tris-acetate, pH 7.6, 5 mM MgOAc, 0.25 mM [beta]-mercaptoethanol and 5 [mu]g/ml BSA), 0.2 mM dNTP, 0.9, 1.8 or 2.7 [mu]M T7-dN
6
primer and 0.1 [mu]g pNL1 plasmid in each case. The reactions were heated at 100oC to denature the DNA for 5 min and cooled to room temperature. Klenow
fragment (10 U) was added to each mixture before incubating them at 37oC for 2 h. Thereafter, to one reaction, the reaction mix was again heated
at 100oC for 5 min and another 10 U Klenow was added for a second round of Klenow
random synthesis. The second reaction had no additions. Excess T7-dN
6
primers in the Klenow-synthesized molecules were subsequently removed through Centricon-100 by washing with 2 ml sterile water twice.
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