John M. S. Bartlett.pdf - Bio-Nica.info

Cloning Gene Family Members 487
Table 1
The Degenerate Nucleotide Alphabet
Letter
Specification
A
Adenosine
C
Cytidine
G
Guanosine
T
Thymidine
R puRine (A or G)
Y pYrimidine (C or T)
K Keto (G or T)
M aMino (A or C)
S Strong (G or C)
W Weak (A or T)
B Not A (G, C, or T)
D Not C (A, G, or T)
H Not G (A, C, or T)
V Not T (A, C, or G)
N aNy (A, G, C, or T)
I
Inosine a
a Although inosine is not a true nucleotide, it is included
in this degenerate nucleotide list because many researchers
have employed inosine-containing oligonucleotide primers
in cloning gene family members.
has been broken up into three parts: Subheading 3.1. describes the designing of the
degenerate primers; Subheading 3.2. describes the PCR amplification with degenerate
primers; and 3. Subheading 3.3. describes the subcloning and DNA sequencing of the specific
PCR-amplified products.
2. Materials
2.1. Design of Degenerate Oligonucleotide Primers
No special materials are required here, except the amino acid sequence to which
the degenerate primers will be designed and a codon usage table (5). If the degenerate
primers are going to be designed according to a family of related amino acid sequences,
these sequences should be aligned using a multiple sequence alignment program. A
degenerate nucleotide alphabet (Table 1) provides a single-letter designation for any
combination of nucleotides. Some investigators have successfully used mixed primers
containing inosine where degeneracy was maximal, assuming inosine is neutral with
respect to base pairing, to amplify rare cDNAs by PCR (10,11).
2.2. PCR Amplification with Degenerate Primers
For all buffers and reagents, distilled deionized water should be used. All buffers
and reagents for PCR should be made up in distilled deionized 0.2-µ filtered water that
has been autoclaved (PCR water) using sterile tubes and aerosol blocking pipet tips to
prevent DNA contamination (see Note 1). All plastic supplies (microfuge tubes, pipet
tips, and so on) should be sterilized by autoclaving or purchased sterile.