Questions
1. Give examples of cases where site-directed mutagenesis can be useful.
The applicatons of site-directed mutagenesis can generall be divided into two
categories:
1. Tools for mapping the functions and properties of a DNA sequence, an
RNA sequence or a protein.
2. Rational design of proteins.
By introducing a specific mutation in DNA and investigating the effect of this
mutation on the DNA sequence or its product, functions and properties may be
deduced. Site-directed mutations may also be introduced in order to tailor the
properties of properties based on theoretical analyses.
2. The DNA-sequence encoding the NNHSYW-motif in algL can also also be
mutated to sequence corresponding to NNASYW. Suggest a QuikChange
primerpair that changes the NNHSYW to NNASYW. The mutagenic primers
should 1) encode the NNASYW-motif in the right reading frame and 2) contain
a new restriction site for identification of the mutaion (find an alternative to
BpmI!). Be careful to only introduce “silent mutations”, except for the
intended H→A-mutation. The length of the primer should be ~45 nucleotides
and the mutations should be as close to each other and as centered in the
primer as possible (…).
The wild-type sequence with amino acid sequence for the correct reading frame:
acctgccgct ggaaaaaacc aacaaccact cctactgggc cgcctggtcg
l p
l e k t
n n h
s y w
a a w s
Histidine (h) in the wild-type sequence should be changed to alanine (a). Alanine
is encoded by all codons starting with gc, i.e. gct, gcc, gca and gcg. In this case, the
simplest approach is to change the first two nucleotides in the histidine codon
from ca to cg, thereby changing the codon from cag to gcc. Furthermore, the
primers should a new restriction site introduced throug a silent mutation, i.e.
without changing the amino acid sequence of the protein. There are several ways
to achieve this. One example is:
acctgccgct Cgaaaaaacc aacaaccact cctactgggc cgcctggtcg
Here, a mutation from g to c has been introduced in nucleotide number 11 from
the left (signified by an uppercase letter). The new codon, ctc, encodes leucine,
just like the original codon, ctg. The underlined sequence is the restriction site of
the enzyme AccBSI [1]. Another exampe is:
acctgccAct ggaaaaaacc aacaaccact cctactgggc cgcctggtcg
This mutation changes nucleotide number 8 from the left from g to a. The codon
is thereby changed from ccg to cca; both of these encode proline. The underlined
sequence is the restriction site of Bse1I [2].
Here, it is chosen to utilise the first example to introduce a restriction site. The
desired mutant sequence then becomes:
acctgccgct Cgaaaaaacc aacaacGCct cctactgggc cgcctggtcg
l p
l e k t
n n A
s y w
a a w s
Mutations that are introduced in the DNA sequence as wall as the resulting
mutation in the protein product are signified by uppercase letters. The
restriction site of AccBSI is underlined. The following mutagenesis primers can
be used to achieve this:
Forward primer: 5’-cctgccgctcgaaaaaaccaacaacgcctcctactgggccgcctg-3’
Reverse primer: 5’-caggcggcccagtaggaggcgttgttggttttttcgagcggcagg-3’
The forward primer contains the desired mutations and is otherwise identical to
the original wild-type sequence. The reverse primer is complementary to the
forward primer and thus contains mutations that are complementary to those
introduced in the forward primer. Both primers have a length of 45 bp.
References
1. http://rebase.neb.com/rebase/enz/AccBSI.html
2. http://rebase.neb.com/rebase/enz/Bse1I.html