Biomedical Engineering Reference
In-Depth Information
a
b
unknown
sequence
adaptor
known sequence (K)
A
U
K1
K2
Specific
product
K2
A
U
K1
Specific
product
1
2
+
1
2
A
K1
Linearized
QC cloning
Vector
A
ns
K2
CS
A
ns
A
Non-specific
products
K2
ns
K2
A
U
K1
Cloned
insert
AA
AK2 K2 K2
Fig.
1
Principle of the QC cloning strategy. (
a
) DNA fragments containing known and unknown fl anking
sequences are amplifi ed by PCR using a primer homologous to an adaptor sequence (primer 1 and sequence
A) attached to the end of the unknown sequence (U) and a primer homologous to a region of the known
sequence (primer 2 and region K2). In addition to specifi c products, PCR amplifi cation can yield nonspecifi c
products (ns) and primer dimers. (
b
) Fragments are cloned via homology between sequences present in both
the insert and the vector: sequences A and sequence K1 (also called the catching sequence, CS). Since primer
2 used for PCR amplifi cation of the insert does not overlap with region K1, nonspecifi c products and primer
dimers cannot be cloned
exclusively to the sequences fl anking the known sequence of
interest, a mixture of specifi c and unspecifi c products can be ampli-
fi ed. This is in addition to other nonspecifi c products such as
primer dimers that can be amplifi ed as by-products in all PCR
amplifi cations (Fig.
1a
). The QC cloning strategy was designed to
clone exclusively the specifi c products from a mix that contains
both specifi c and unspecifi c products. QC cloning is a ligation-
independent cloning strategy that uses homology between
sequences present in both the vector and the insert. One end of the
linearized QC cloning vector is homologous to the adaptor
sequence used for amplifi cation of the insert (Fig.
1b
). In contrast,
the other end of the linearized vector does not have homology to
the other primer used for amplifi cation of the insert (binding to
region K2), but rather has homology to a sequence of the known
region nested and non-overlapping with the primer used for ampli-
fi cation (region K1). Since this sequence is only present in specifi c
products, only these can be cloned.
Cloning of specifi c sequences using QC cloning therefore
requires preparing a specifi c cloning vector for each new sequence
that needs to be cloned. The insert is cloned in the QC vector
using a one-pot incubation with T4 DNA polymerase followed by
direct transformation of the incubation mix in competent cells.
Individual clones can then be sequenced with a primer in the
known region or with vector primers.
