Efficient Algorithm for Primer Design

PCR is one of the most popular technique that enable amplify specific region of the genome. It allows performing variety of analyses and application including DNA cloning for sequencing; functional analysis of genes; the diagnosis of hereditary diseases. Primer design is one of the most fundamental in PCR methods. Many different parameters need to be taken into account. There is many commercial program, some of them are online available, for the primer design. In this paper we present an algorithm which is not located in analyzing large sequence but for infrequent users.


INTRODUCTION
In 1983, biochemist KaryMulis invents a technique that called polymerase chains reaction (PCR) to amplify a single or few copies of piece of DNA. This technique which brings Nobel Prize in Chemistry to him enables to generate millions of copies of a particular DNA sequence. It allows performing variety of analyses and application including DNA cloning for sequencing; functional analysis of genes; the diagnosis of hereditary diseases ( The methods, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic reaction of the DNA, relies on thermal cycling. Short DNA fragments that are complementary to the target region along with DNA polymerase are key components to perform repeated amplification (Yuryev, 2002) Since the technique relies on thermal there is need to employ a heat stable DNA polymerase. It is naturally exist in bacterium Thermusaquaticus (Boyle, 2009)  Abstract PCR is one of the most popular technique that enable amplify specific region of the genome. It allows performing variety of analyses and application including DNA cloning for sequencing; functional analysis of genes; the diagnosis of hereditary diseases. Primer design is one of the most fundamental in PCR methods. Many different parameters need to be taken into account. There is many commercial program, some of them are online available, for the primer design. In this paper we present an algorithm which is not located in analyzing large sequence but for infrequent users.
In 1983, biochemist KaryMulis invents a technique that on (PCR) to amplify a single or few copies of piece of DNA. This technique which brings Nobel Prize in Chemistry to him enables to generate millions of copies of a particular DNA sequence. It allows performing variety of analyses and application DNA cloning for sequencing; functional analysis nosis of hereditary diseases (Kary, 1993).
The methods, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic reaction of the DNA, relies on thermal cycling. Short DNA fragments that are complementary to the target region y components to (Yuryev, 2002).
Since the technique relies on thermal there is need to employ a heat stable DNA polymerase. It is naturally exist bacterium Thermusaquaticus (Boyle, 2009  PCR is one of the most popular technique that enable amplify specific region of the genome. It allows performing variety of analyses and application including DNA cloning for sequencing; the diagnosis of hereditary diseases. Primer design is one of the most fundamental in PCR-based methods. Many different parameters need to be taken into account. There is many commercial program, some of them are this paper we present an algorithm which is not located in analyzing large sequence but chemically synthesized oligonucleotides with a short length of about 20 base pairs. Since the primer is crucial component for the PCR application, primers needs to be design very carefully with considering some important mathematical aspect. Because the primers are functioning in pairs it has to designed as so called forward and reverse primers. They have designed as they will be extended towards each of cover the given target PCR begins with approximately 95 C0. This step called denaturation. In this step the double strandedDNA converted into single stranded DNA. In the second step of the PCR, annealing step, primers hybridize to the sequence, this step has temperature ranging between 45-62 led extension, in this step intermediate temperature about 72 0C applied to extend the hybridized primers in both directions. This all steps are called one cycle and each of the PCR application typically 1.1.

Principle of Primer Design
After the usage of PCR in research and clinic, designing primer becomes critical. There is many software and desktop programs, even some of them are online, that are aimed to design a primer in efficient way. There are several criteria to design a efficient primer and these criteria`s are directly effecting the efficiency of PCR.
• GC contents: No large GC rich or deficient content.
• Secondary structure in primer: no hairpin secondary structure.
To perform PCR consistently in every cycle Melting Temperature of the primers must be similar. Generally programs use the original nearest neighbor method (Breslauer, 1986) to determine Tm. Beside the nearest neighbor method, programs also use same method with empirically determined thermodynamics values to determine Tm with the DNA target according to the following equation (Sugimoto, 1996). The other one of the important criteria is G/C content. Since G-C content contains three hydrogen bonds whereas an A-T content has two. Three hydrogen bonds make the G-C content stronger so the melting temperature needs to be increase in order to break these bounds. Generally, G/C content of the assumption is to arrange GC content between 40 and 60% (Lowe, 1990).
Self-complementation is also one of the biological facts that must be avoided when designing primers. Primers enable either the formation of secondary structure in the single stranded oligonucleotides, or binding another copy of itself (Haas, 1998). This self-complementation called primer dimer. Both of the complementation must be considered when designing the primer.
Primer length is a criterion which is directly related to the uniqueness. Also hybridization stability is related to the length of the primer. While increasing the length of primer cause mismatch tolerance, shorter primer has specificity problem. Especially according to the real PCR data, short primer don't show specificity to the target region of the sequence and it manipulates the result of PCR.

Algorithm in primer design
Software that is developed for biological application gives insight to the field of bioinformatics. There are many different programs for designing primers and some of them are available online. These programs designed by some companies that are engaged in bio software development include: AlkamiBiosystems, Molecular Biology Insights, PREMIER Biosoft International, IntelliGenetics Inc., Hitachi Inc., DNA Star, Advanced American Biotechnology and Imaging. Some scientists have also developedAlgorithm and computer programs for various purposes of primer design (Abd-ElsalamKamel, 2003).
In 2012 BLAST introduce a new software toll called Primer BLAST. With this program BLAST claim that program design target-specific primers. It uses global alignment algorithm to ensure a full primer target. The very useful features of the program is checking the preexisting primers and able to place primers based on exon intron location and excluding single nucleotide polymorphism sites in primers. Primer BLAST uses Needleman-Wunsch algorithm to perform pairwise alignment through the entire length of the sequences. This program was implemented using the C++ toolkit and the primer3 C programming interface (Jian Ye et al, 2012 (Wu, 1991).
Technically it is not difficult to arrange the primer length but the other variable needs to be correlated with it.

Melting temperature
In molecular biology oligonucleotide melting temperature is important for many techniques. For these reason several formulas have been developed to estimate melting temperature. One of the very popular one is nearestneighbor model. Generally it gives highest accuracy for Tm prediction. İt is not certain for adjusting these models for the effects of reagents commonly used in PCR. Ahsenet all design an experiment that investigate 475 matched or mismatched target-probe duplexes. This data set was used to evaluate the contributions of[Mg21], [dNTPs], and [DMSO] in N-N calculations. This study succeeded to predict accurate melting temperature with using N-N calculation or empirical formulas (Wallacem, 1979).

GC Content:
Strength if annealing can be determined with looking GC content of primer since it is highly depends on GC content. Primers that have less than 50% GC content should be extended beyond 18 bases to keep the melting temperature above the recommended lower limit of 50 0C. Rychliket all describe the dissociation temperature. Since some algorithm are based solely on length of the oligonucleotide but reliable only when hybridizations are performed in some specific solution (Dieffenbach,1995). More practical way to determine the dissociation temperature has been described by Sugget all as in the following formula : nearest neighbor thermodynamic parameter. OLIGO is described as follows [17]: where AH and AS are the enthalpy and entropy for helix formation, respectively, R is molar gas constant [1.987 (cal/°C x mol)], and C is concentration of the probe.

3'-End Sequence:
Mis-priming need to be minimizes or completely be avoided, if possible, to perform good PCR. Kwok and his friends investigate the effects of primer-template mismatches on PCR. Since the 5` ends and 3` ends should be sticky. These ends needs to be rich in GC, and sites could potentially anneal at multiple sites in the template. They investigate whether the effects of mismatches on PCR were symmetrical. For instance, would a G: T, primer-template, has the same effect on PCR as T: G, template-primer mismatches. They figure out that there is a sympatric effect of mismatches and they indicate that could be due to flanking sequences (Kwok,1990).

METHODS
The algorithm designed with considering 4 criteria. Each of the criteria is listed as follow; The DNA nucleotide sequence is taken as input of character array. In this algorithm both end of the sequence are considered as a target not the entire DNA sequence; to illustrate in this algorithm. The first 100 nucleotide of the sequence are taken as possible target. User allowed enlarging the target area. The length of the sequence is determined by the user, which can be range between 18-25. In this basis the number of GC are counted and their proportion has taken to the entire bases. Since the 50 % has strong certainty, it is counted in range between 45% and 55%. The basis which satisfies this percentage, they are taken as possible candidate of primers.  There is many different approach to develop a good algorithm for designing PCR primer. In this paper many biological aspect was considered to design an efficient algorithm to design proper primer to increase the efficiency of PCR. Algorithm has been run successfully with considering the mentioned biological facts. This algorithm allow user to adjust the target sequence manually to protect the functional parts of the sequence. However, there is need to use the primer that are already designed. Since BLAST is using its own database which contains entire genome of many different species, it has a big advantage over the other algorithms that are designed for the same purpose. There is need for more accurate and powerful program to design efficient primer, since the biological knowledge are expending day by day.