1.
Propose which amino acids and which nucleotides should be included in a final
product that contains cysteines 256 and 259, but does not contain cysteine 250.
Define the appropriate sequences by numbers (for example, amino acids 1-10 and
nucleotides 1-30)
Answer:
The simple amino acids will be suitable to exchange the codon (UUC) of Cystine
at 250th position through the technique known as site-directed mutagenesis. Two
of the best examples of these amino acids are alanine and glycine, both of
which have no reactive group. If the current amino acid is replaced with
alanine and glycine, which have their own ionization and belong to reactive
groups like NH3+ and –OH, then bonds will easily be formed, and this will help
change the shape of Fc protein.
2.
Design a forward (sense) primer to amplify the DNA sequence you have listed in
Question #1. Calculate the %GC content, the melting (Tm) temperature, and the
annealing (Ta) temperatures for the primer. Be aware that this primer must work
together with the primer you report in Question #3.
Answer:
For primer (1-21 nucleotides), a forward sense or
primer to amplify the DNA sequence is as follows:
3’-AGGTGGTTGGGTAGCCAGAAG -5’
·
LENGTH = 21
·
C+G% = 57.1
·
Molecular weight = 6672.02
·
Tm = 56.3 °C
3.
Design a reverse (antisense) primer to amplify the DNA sequence you have listed
in Question #1. Calculate the %GC content, the melting (Tm) temperature, and
the annealing (Ta) temperatures for the primer. Be aware that this primer must
work together with the primer you report in Question #2.
Answer:
In order to amplify the DNA sequence, the sequence of reverse (antisense) primer is given below:
5’-TCATTTACCCGGGGAGAGGCT-3’
·
LENGTH = 21
·
C+G% = 57.1
·
Molecular weight = 6542.91
·
Tm = 56.3 °C
4.
What is the size of the PCR product predicted from a reaction using the primers
in Questions #2 and #3 with an IgG1 cDNA template?
Answer:
The most suitable size of the PCR product from a reaction of the (antisense) primer and the primer (1-21 nucleotides) will be 75–150 bp.
5.
Suppose you re-design your primers to include a XbaI restriction site on the
forward primer and a HindIII restriction site on the reverse primer. These
primers will introduce a XbaI site onto the 5’ end of the insert, and a HindIII
site onto the 3’ end of the insert, to allow directional cloning into your
plasmid. When the clone described above has been successfully isolated, you
will have a new plasmid that carries the appropriate human IgG1 Fc sequence
inserted at unique XbaI and HindIII sites. To create an expression vector that
will direct production of fusion proteins, you plan to insert the gene sequence
for your protein of interest into this plasmid upstream of the Fc sequence
using non-directional cloning at the XbaI site. (Drawing this should help you
understand the strategy.) In addition to creating XbaI restriction sites at
both the 5’ and 3’ ends of the insert for your protein of interest, what
important feature or codon must be included or designed into the primers used
to make your construct to ensure protein production? Hint: it will be at the 5’
end of the insert sequence, just after the XbaI site.
Answer:
Besides creating XbaI restriction sites at both the 5’ and 3’ ends of the
insert for the protein of interest, the codon or important feature that needs
to be included or designed into the two primers used to make the your construct
to ensure protein production is 4.9.
