190
KLAUS DEICHMANN
from M13 have been extensively modified. They contain a so-called multiple cloning region (MCR), in which the inserts are cloned into defined,
unique restriction sites. In addition they contain apart of the beta-galactosidase gene of E. coli (lacZ gene), allowing by color selection a destinction between vectors bearing an insert from those lacking an insert.
All have been constructed in pairs differing by the order of restriction
sites in the MCR (e.g. M13mp18/M13mp19). This allows direct sequencing ofboth the complementary strands of the DNA insert. The fragment
to be sequenced can be ligated into the RF form, and single-stranded
DNA can be recovered from the supernatant after extrusion. The extruded DNA can be used directly in sequencing reactions.
• pUC plasmids
These plasmids (Vieira and Messing 1982; Sambrook et al. 1989) are
widely used in sequencing reactions. They contain an MCR as described
for the M13 phages, the same E. coli lacZ gene, and, in addition, aß Iactamase gene, confering ampicillin resistance to infected host cells. Once
the host cells have been transformed with the insert-containing plasmid,
the recombinant plasmid will replicate to a high copy number. Prior to
the sequencing reactions, the plasmids are denatured by heat or alkali,
yielding single-stranded DNA. However, in order to achieve good quality
of the template DNA purification of the plasmids is recommended, e. g.
by use of ion exchange chromatography (see Chap. 15, Subprotocol 5).
• Inserts
It is possible to read more than 1000 bp on one gel using special gel-casting and sequencing techniques. However, routinely, 400 to 600 bp can be
read from the gel.
• Primers
Fortunately, the sequences flanking the MCR in most vectors are similiar
so that a small set of primers is sufficient to perform sequencing on most
plasmids. Theseare called universal primers. They are typically 15 to 29
nucleotides, as a shorter length carries the risk of false annealing and a
Ionger length increases the risk of secondary structure formation of the
primers. N early all primers are commercially available.
• DNA polymerases
Four different polymerases are used for sequencing:
- Klenow fragment of E. coli DNA Polymerase I ( Sanger et al. 1977), the
"classical" sequencing enzyme. The Klenowfragment lacks the 5'- to -3'
exonuclease activity of native DNA polymerase I and provides faithful
synthesis of a complementary strand from a single-stranded template.
KLAUS DEICHMANN
from M13 have been extensively modified. They contain a so-called multiple cloning region (MCR), in which the inserts are cloned into defined,
unique restriction sites. In addition they contain apart of the beta-galactosidase gene of E. coli (lacZ gene), allowing by color selection a destinction between vectors bearing an insert from those lacking an insert.
All have been constructed in pairs differing by the order of restriction
sites in the MCR (e.g. M13mp18/M13mp19). This allows direct sequencing ofboth the complementary strands of the DNA insert. The fragment
to be sequenced can be ligated into the RF form, and single-stranded
DNA can be recovered from the supernatant after extrusion. The extruded DNA can be used directly in sequencing reactions.
• pUC plasmids
These plasmids (Vieira and Messing 1982; Sambrook et al. 1989) are
widely used in sequencing reactions. They contain an MCR as described
for the M13 phages, the same E. coli lacZ gene, and, in addition, aß Iactamase gene, confering ampicillin resistance to infected host cells. Once
the host cells have been transformed with the insert-containing plasmid,
the recombinant plasmid will replicate to a high copy number. Prior to
the sequencing reactions, the plasmids are denatured by heat or alkali,
yielding single-stranded DNA. However, in order to achieve good quality
of the template DNA purification of the plasmids is recommended, e. g.
by use of ion exchange chromatography (see Chap. 15, Subprotocol 5).
• Inserts
It is possible to read more than 1000 bp on one gel using special gel-casting and sequencing techniques. However, routinely, 400 to 600 bp can be
read from the gel.
• Primers
Fortunately, the sequences flanking the MCR in most vectors are similiar
so that a small set of primers is sufficient to perform sequencing on most
plasmids. Theseare called universal primers. They are typically 15 to 29
nucleotides, as a shorter length carries the risk of false annealing and a
Ionger length increases the risk of secondary structure formation of the
primers. N early all primers are commercially available.
• DNA polymerases
Four different polymerases are used for sequencing:
- Klenow fragment of E. coli DNA Polymerase I ( Sanger et al. 1977), the
"classical" sequencing enzyme. The Klenowfragment lacks the 5'- to -3'
exonuclease activity of native DNA polymerase I and provides faithful
synthesis of a complementary strand from a single-stranded template.