Biomedical Engineering Reference
In-Depth Information
a
b
T095 (IgG
κ
)
PCR products
T095 (IgG
κ
)
Colony PCR
KC2F
in pKC2
1500
GC2F
in pGC2
1500
500
500
1500
1500
GC2N
in pGC2
KC2N
in pKC2
1.5 kb
0.5 kb
500
500
* * * *
*
* *
GC3F
in pGC3
1500
KC3F
in pKC3
1500
500
500
Water
control
KC3N
in pKC3
1500
500
Size of desired, full-length
inserts (smaller inserts
are truncated, but specific)
PCR products selected
for cloning
*
Fig.
3
QC cloning of immunoglobulin fragments from patient sample T095. (
a
) PCR products containing the
variable region (unknown region) and a fragment of the constant region (the known region) of immunoglobulin
Kappa light chains amplifi ed from non-Hodgkin lymphoma biopsy sample T095. The products were amplifi ed
using a G-tail anchor primer (binds the adaptor sequence) and immunoglobulin constant region-specifi c prim-
ers (GC2F, GC3F, etc.). The PCR products indicated by an
asterisk
were cloned in corresponding QC cloning
vectors. (
b
) Randomly chosen clones from each cloning experiment were analyzed by colony PCR using vector
primers. PCR products were separated on a 1 % agarose gel supplemented with ethidium bromide and visual-
ized under UV light. The expected insert size is indicated by a
dashed line
pUC19 sequences. Since the same adaptor sequence can be
used for amplifi cation of different sequences of interest, this
primer can be reused for different cloning experiments. The
second primer, primer 2 (
gccctgggctgcctggtcaaggactacttcccc
cgtctccgggagctgcatgtg), contains an extension that is homolo-
gous to region K1 (italics). Since this region is specifi c to each
sequence of interest, primer 2 needs to be modifi ed for each
new cloning project. PCR amplifi cation performed using prim-
ers 1 and 2 and pUC19 as a template results in a 2.7 kb back-
bone fragment. The PCR product just needs to be
column-purifi ed and can be used directly for QC cloning. Since
it is a linear fragment, the PstI digestion step described for lin-
earization of QC cloning vectors is not needed anymore.
2. We have investigated the length of catching sequences suitable
for QC cloning. A CS of 12-15 nucleotides is suffi cient for
QC cloning (as is also known for other LIC strategies [
12
]).
However, a polymorphism of one nucleotide in the region of
the amplifi ed insert homologous to a 12-nucleotide CS is
likely to prevent cloning of this insert. In contrast, using a
50-nucleotide CS will allow homologous sequences contain-
ing one or a few mismatches to be cloned (as long as a stretch
of 12-15 consecutive nucleotides is present in both the insert
and the catching sequence).
3. BpiI is a type IIS restriction enzyme whose cleavage site is
located outside of its DNA recognition site. As a result, ligation
