Introduction
Molecular biology tools include polymerase chain reaction (PCR) which requires a mRNA template or a cDNA template, polymerase enzyme and a primer; mainly an oligonucleotide primers. The design of the best primer is important for the success of many molecular biology techniques such as polymerase chain Reaction, hybridization, sequencing of DNA, and Micro Array techniques. Primers have also been exploited in separation and isolation of DNA using special Southern blotting technique. However, the primer used in this case is a radio labeled probe that binds very well with DNA nucleotides. Primers are thus an essential tool for PCR process. They are short single stranded nucleotide sequences ranging from 16-25 nucleotide bases. Above 20 nucleotide sequences, “the primer allows a single mismatch which is the cause of genetic mutations therefore the best primer needs to be precise” (Thieman and Palladino, p 85).
Primers Formation and Justification
The nucleotide sequences given of the xylanase gene of the four micro-organisms have the following primer sequences as shown. It should be noted that the primers are read from the 5’end to the 3’ end. That is, the forward and backward primer which are either at the 5’ end or 3’end respectively. The 5’end primers are obtained by direct reading of nucleotide sequence of each micro-organism in the provided sequences. The 3’ end primers are obtained from the complementary strand of the nucleotide sequences provided which acts as a template and the primer is read from the 5’ to 3’ end. In this case, I have chosen on 18 nucleotide sequences as the best primer sequence as explained earlier.
The primers for the microorganism HUMICOLAXYN2 are as follows.
At the 5’ end: 5’ATGGTCGACCTGCAGGCG 3’ while the 3’ end primer is 5’AGTCTGGACATAGATGTC3’.The primers for the micro-organism TRC3ΘXYN1, at the 5’ end: 5’ATGGTCTCCTTCACCTCC3’ while at the 3’ end, the primer is
5’TTAGCTGACGGTGATGGA3’.
The primers for the Microorganism TRC3ΘXYN2 are; at the 5’ end 5’ATGCCCACAGGCCTCGAG3’ while at the 3’ end; 5’ GTTGCTGACACTCTGTGA3’.
The primers for the DICTYXYNB are; at the 5’ end; 5’ATGTTTCTTAAAAAACTT3’ while at the 3’ end; 5’AGTATTTTGTGTTATATT3’.
Having the primer and the template, DNA amplification of our gene of interest can be done. This can be further used for Chromosomal Walking PCR to isolate the rest of the gene. There are various techniques of chromosomal walking PCR, “this includes: Inverse PCR, and tail PCR which can either the 5’ RACE (Random isolation of cDNA ends) or 3’RACE” (Saikia, p 127). To obtain the 5’sequence (5’RACE) the first strand of the sequence is tailed with a string of dATPs or dGTPs using terminal tranferase. A universal sequence is then used verses the specific primer in PCR. For the 3’ sequence, a specific primer is designed and used against a universal Primer comprised of an oligo (dT) sequence with an additional 5’ bases, generally restricted sites for increased stability.
Features/Characteristics of a Primer
Primer base sequences are supposed to be 16-25 bases in length, the G+C composition of the primer sequence should range between 40-60%. This GC pairing is stronger than AT pairing, thus, it enhances primer stability. The 3’ end of a primer should end in a C or G or CG or GC. This increases the priming efficiency as it prevents the breathing of the primers end; that is to prevent dimerization. The annealing temperature of the primer is also critical as it is affected by both the length and the sequence of the primer. “The annealing temperature (Ta) is the temperature at which the primer binds optimally and specifically to the template” (Meyers, p 547). It should be as high as possible in order to prevent random binding to other sequences and also reduce the chances of cyclization during the PCR. Ta is taken to be less by 15 of the melting temperature (Tm).Tm of a primer is calculated by ascribing a temperature of2oC for every A or T in the sequence and 4oC for every G or C. Primer sequences should also be checked for self complementation, formation of hair pin loops and ability of hybridize preferentially with each other rather than the template.
Amino Acid Reverse Translation
“The amino acid sequence can be reverse translated to generate a DNA sequence which in turn can be amplified to generate the nucleotide sequences of our genes of interest, in this case the xylanase gene. “This mostly represents the non degenerate coding sequences of DNA nucleotide” (Fitch, p 74). This amino acid sequences are reverse translated using a codon table and the variable bases should be highlighted. The number of variable bases should be kept to a minimum. Codon usage does vary between different organism and this can be further exploited to reduce the redundancy.
Work cited
Fitch, Patrick J. An Engineering Introduction to Biotechnology. Tutorial texts in optimal Engineering. Philadelphia: Spie press monograph. 2002. Vol 55: issue 55.
Meyers, Robert A. Molecular biology and biotechnology: A comprehensive desk. New Jersey: Wiley-VCH, 1995.
Saikia, Ratul. Microbial Biotechnology. India: New India publishing company. 2008.
Thieman, William J., Palladino, Michael A. Introduction to Biotechnology. 2nd Ed. San Francisco CA: Pearson Benjamin-Cummings Publishing Company. 2009.