Minimum GC-Rich Sequences for Overlap Extension PCR and Primer Annealing - DNA Cloning and Assembly Methods - page 170

a

Gene 1 Gene 2

SKL

SKL

Flag Flag

RE RE

6His 6His

SKL SKL

Flag Flag

RE RE

6His 6His

b

HRS1 9C

Marker gene

3CG9

HRS2

HRS1 HRS2

Fig. 5 Application of minimum annealing sequences for the addition of various
sequences. ( a ) The minimum annealing sequences are generated to the end of
various DNA fragments by PCR with primers containing additional minimum
9-nucleotide sequences ( arrowheads ). The annealing sequence can be used
together with a set of primers containing desired additional sequences, such as
SKL peroxisomal targeting sequence, FLAG tag, restriction enzyme sites (RE),
and 6His affi nity purifi cation tag for gene manipulations. These primers or the
other additional sequence primers can be used universally for their addition to
any genes through the minimum annealing sequences. ( b ) The minimum anneal-
ing sequences can be used simultaneously if they are not complementary. For
example, a transformation marker gene is amplifi ed with primers containing
homologous recombination sequences (HRS) through 9C and 3CG9 annealing
sequences for gene targeting in yeast ( see Note 13 )

to any templates with the same minimum annealing sequences
(Fig. 5 , see Note 13 ).
To show the effi ciency and accuracy of the annealing of the
minimum sequences, in-frame addition of a functional peptide
coding sequence is demonstrated in this section.
As an example of in-frame addition of a peptide sequence,
C-terminal peroxisome-targeting signal SKL (Ser-Lys-Leu) is selected
[ 16 ]. First, yEmRFP (yeast-codon optimized monomeric RFP) [ 11 ]
is amplifi ed with primers containing minimum annealing sequences.