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For the Tutorial Programme in Proteomics
High-Throughput Cloning and Expression Library Creation for
Functional Proteomics - Supplementary Material
Basic concepts – Supplementary Material
1.
Main enzymes used in cloning methods
DNA polymerase. The synthesis of a new strand of DNA is a process mediated by a
DNA polymerase. It requires the use of a template, which can be a DNA or RNA molecule, the
latter resulting in the complementary copy of the mold. DNA-dependent DNA polymerase was
first isolated in Escherichia coli in 1958 (Lehman et al 1958) and in 1986 the Polymerase Chain
Reaction (PCR) was developed (Mullis et al 1986, Saiki et al 1988). PCR is an in vitro reaction
that allows the copy of a single DNA molecule into millions, an extremely important feature for
DNA cloning. In a PCR reaction, short sequences of DNA, called primers, target specific
sequences and .then provide a starting point for the amplification. Since the DNA amplification
only occurs in the 5
3’ orientation and the two strands of the DNA are anti-parallel, two
primers are used in the PCR reaction: one that aligns with the end of the target sequence in the
5’-3’ strand of the template, and another one that aligns with the beginning of the target
sequence in the complementary strand (3’-5’). Each cycle, the template DNA is denaturated, the
primers align with the template and the DNA is amplified, exponentially amplifying the region
flanked by the two primers (Supplementary Figure 1A).
RNA-dependent DNA polymerases, also known as transcriptases, are used to copy
the information from RNA molecules into DNA, referred as copy-DNA (cDNA) (Supplementary
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Figure 1B). cDNA synthesized from gene transcripts can be used as templates for PCR
reactions and allow the cloning of the transcript into vectors.
Restriction enzymes type II and ligases. These two sets of enzymes have
complementary activity, restriction enzymes work as “scissors” capable of identifying and
cleaving specific DNA sequences (Kelly and Smith 1970, Smith and Wilcox 1970), whereas
ligases re-join two DNA strands and reconstitute the phosphodiester bonds (Weiss and
Richardson 1967). Restriction enzymes provide bacteria with protection against exogenous
DNA from viruses and other bacterial strains, working as a rudimentary immune system (Arber
1965, Roberts 2005). To cleave the DNA, restriction enzymes make two incisions, one in each
DNA strand, and depending upon the relative position of the two incisions the product can have
a blunt end or, with staggered cuts, overhanging ends (Williams 2003) (Supplementary Figure
2). For DNA cloning, the vector and the gene of interest have to be digested with restriction
enzymes that possess compatible products to allow the strands to come together in perfect
alignment, for subsequent ligation.
Recombinases. Nucleic acid recombination is a process in which DNA fragments
are exchanged between two DNA molecules and it is mediated by enzymes called
recombinases. These enzymes have the intrinsic activity to cut and ligate DNA fragments, within
a single complex. Recombination was originally described in maize by Barbara McClintock in
1931 (Creighton and McClintock 1931, McClintock 1931), however it took five decades for the
first recombinase and its precise mechanism of action to be elucidated (Abremski and Hoess
1984, Hoess and Abremski 1984). Recombinases may present distinct properties regarding the
mechanism of action, substrate preferences and types of products generated. For DNA cloning
it is important that the recombination happens precisely in specific sequences and generates
consistent products every time, without adding or removing any nucleotide during the ligation of
the two DNA molecules. Enzymes with those properties are called site-specific recombinases.
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Depending on the configuration of sequences recognized by these enzymes, the recombination
reaction can be unidirectional, between distinct DNA sequences (Supplementary Figure 3A), or
bidirectional, among identical sequences (Supplementary Figure 3B). This process is largely
independent of the sequence to be cloned, making it suitable for high-throughput cloning
projects.
2.
Vector features
In functional genomics the term plasmid refers to a circular double stranded DNA
with the ability to replicate within the cell, independently of the genomic DNA. The process to
introduce a plasmid (clone or empty vector) into bacteria is called transformation and it is
commonly used for the amplification and the isolation of plasmids. In eukaryotic cells, the
introduction of a plasmid into cells is designated transfection (if it is done chemically) or
transduction (if it is done using viral delivery).
The basic elements common to all vectors include: the replication site, antibiotic
resistance, and cloning site (Supplementary Figure 4).
The replication site, or origin of replication, comprises a nucleotide sequence that
can be recognized by the cellular DNA replication machinery as a start point for the synthesis of
the new copy (Nagata and Meselson 1968). Different organisms recognize distinct replication
sites, therefore the vector must have the origin of replication appropriate for the host cell of
choice (Errico and Costanzo 2010). Once in the cell, the vector is copied multiple times and
shared among daughter cells after the cell divides. Alternatively, some vectors, usually the ones
derived from virus, are integrated into the host genome and passed to the progeny cells as part
of the genome.
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Selection markers are genes that alter the proliferation capabilities of the host cell.
The purpose of these markers is to provide growth differences between cells with and without
the plasmid, enriching the cell population for the recombinant cells of interest. The selection can
be positive or negative; meaning that cells carrying the plasmid will survive or die, respectively.
Positive selection is usually accomplished by the use of an antibiotic in the growth media and a
gene, coded by the plasmid, that makes the bacterium carrying the gene resistant to the drug. A
classical example is ampicillin and beta-lactamase gene, usually referred as bla gene or AmpR
(Ampicillin Resistance). Recombinant cells expressing bla can grow in media containing
ampicillin, while the parental cells, without the plasmid, are sensitive to the antibiotic and cannot
proliferate. Negative selection can be accomplished by the use of toxic genes, such as ccdB
(DNA gyrase inhibitor) and barnase (ribunuclease), as the selection marker. Once expressed,
the toxic gene kills the host cell. This type of selection is important to avoid the growth of cells
carrying by-products of the cloning process. Negative selection markers can be propagated in
special strains that are resistant to them.
Cloning sites. Vectors also have specific sequences to allow the cloning of the
insert of interest. Depending on the cloning methodology used, those sequences can be sites
recognized by restriction enzymes or recombinases. Restriction sites for many distinct enzymes
are often present in tandem in a region referred as multiple cloning sites (MCS).
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3. References
Abremski K, Hoess R (1984). Bacteriophage P1 site-specific recombination. Purification and properties
of the Cre recombinase protein. J Biol Chem 259: 1509-1514.
Arber W (1965). Host-controlled modification of bacteriophage. Annu Rev Microbiol 19: 365-378.
Creighton HB, McClintock B (1931). A Correlation of Cytological and Genetical Crossing-Over in Zea
Mays. Proc Natl Acad Sci U S A 17: 492-497.
Errico A, Costanzo V (2010). Differences in the DNA replication of unicellular eukaryotes and
metazoans: known unknowns. EMBO Rep 11: 270-278.
Hoess RH, Abremski K (1984). Interaction of the bacteriophage P1 recombinase Cre with the
recombining site loxP. Proc Natl Acad Sci U S A 81: 1026-1029.
Kelly TJ, Jr., Smith HO (1970). A restriction enzyme from Hemophilus influenzae. II. J Mol Biol 51: 393409.
Lehman IR, Bessman MJ, Simms ES, Kornberg A (1958). Enzymatic synthesis of deoxyribonucleic acid.
I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J Biol Chem
233: 163-170.
McClintock B (1931). The Order of the Genes C, Sh and Wx in Zea Mays with Reference to a
Cytologically Known Point in the Chromosome. Proc Natl Acad Sci U S A 17: 485-491.
Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H (1986). Specific enzymatic amplification of DNA
in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51 Pt 1: 263-273.
Nagata T, Meselson M (1968). Periodic replication of DNA in steadily growing Escherichia coli: the
localized origin of replication. Cold Spring Harb Symp Quant Biol 33: 553-557.
Roberts RJ (2005). How restriction enzymes became the workhorses of molecular biology. Proc Natl
Acad Sci U S A 102: 5905-5908.
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT et al (1988). Primer-directed enzymatic
amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491.
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Smith HO, Wilcox KW (1970). A restriction enzyme from Hemophilus influenzae. I. Purification and
general properties. J Mol Biol 51: 379-391.
Weiss B, Richardson CC (1967). Enzymatic breakage and joining of deoxyribonucleic acid, I. Repair of
single-strand breaks in DNA by an enzyme system from Escherichia coli infected with T4
bacteriophage. Proc Natl Acad Sci U S A 57: 1021-1028.
Williams RJ (2003). Restriction endonucleases: classification, properties, and applications. Mol
Biotechnol 23: 225-243.
Figures Legends
Supplementary Figure 1 - Polymerase Chain reaction and cDNA synthesis.
DNA Polymerases are largely employed in cloning techniques. (A) DNA-Dependent DNA
polymerase is used in PCR reactions for the amplification of the desired insert. Every cycle is
composed of a denaturation step, followed by annealing of the primers and extension (DNA
synthesis). After 20-30 cycles, millions copies of the sequence flanked by the primers is
obtained. (B) RNA-dependent DNA polymerase is used to create a DNA copy (cDNA) of the
mRNA.
Supplementary Figure 2 - Restriction enzymes and ligases. Type II restriction
enzymes cut the DNA at specific recognition sequences (often palindromes) creating blunt ends
(A) or cohesive ends (B), depending on the relative position each strand of the DNA is cleaved.
This reaction can be reversed by the action of the ligases, enzymes that ligate two DNA
molecules by reforming the phosphodiester bond.
Supplementary Figure 3 – DNA Recombinases. Site-specific recombinases have
the combined activities of a restriction enzyme and a ligase. They are capable of identifying
specific sequences in the DNA and exchanging those sequences between two distinct DNA
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molecules. The recombination can be: (A) unidirectional, when distinct sites are used, or (B)
bidirectional, using identical sites.
Supplementary Figure 4 – Vector Features. Vectors are circular DNA molecules
capable of replicating independently of the genomic DNA in bacteria, using their origin of
replication (ori). In biotechnology, they usually carry a promoter to drive the expression of a
selection marker, for example beta-lactamase that confers resistance to ampicillin. Cloning sites
are also added to facilitate the insertion of the gene of interest. Depending on the sequence, the
cloning site can be recognized by restriction enzymes or recombinases. In addition to these
features, expression vectors also include a promoter to drive the transcription of the insert and
often a fusion tag in frame with the coding sequences.
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