Protien Essay Example | Topics and Well Written Essays - 1000 words

Protien - Essay Example Due to presence of a high degree of palindromic sequences changes were made in the 3rd codons for R and A, and the 1st codon for L based on the second priority codon preference. The changes reduced the chances of self-annealing and formation of secondary structures. The new gene sequence was thus: The next step was to choose the restriction enzymes for cutting out the complete gene after it has been cloned to the TOPO vector. NdeI, which cleaves the sequence CA/TATG was chosen for the 5’ end while BPu1102 which cuts GC/TGAGC was selected for the 3’ end. Both restriction enzymes do not have restriction sites in the chosen vector (but are present in the expression vector which will be used for protein expression later). GCGC nucleotides are added before and after the NdeI and BPu1102 sites respectively to act as primer initiation sites for PCR of the final gene sequence which is the following: 2. The PCR-based methods for gene synthesis normally require a DNA template, which is not available for designed peptides, for error-free amplification. To reduce error, nucleotide stretches of the optimized gene sequences are synthesized and ligated to complementary sequences followed by PCR amplification (Tsuchiya, Morioka, Shirai, Yoshida, & Inumaru, 2006) (Young & Dong, 2004). These procedures result in different gene fragments that have errors in the sequences. Further cloning, purification and sequencing for the desired gene sequence is expensive and time-consuming. In this study, the gene will be synthesized using circular assembly amplification, a new technique in gene synthesis that removes error sequences and increases the probability of getting accurate sequences (Bang & Church, 2008). Here, a mixture of short complementary oligonucleotides (~ 50bp), that are designed with overlaps to allow complementary coupling or annealing, generates circular DNA. This is followed by exonuclease treatment to remove