Features of primer design for constitutive gene in reverse transcription polymerase chain reaction

Present-day science must base on molecular biology methods to conduct detailed and multifaceted researches. Biochemical, morphological, biophysical and other methods are not enough for description and interpretation of phenomena and mechanisms that occur in a plant cell. The polymerase chain reaction (PCR) is an integral part of many molecular investigations. The PCR is the basis of many research methods, in particular, of reverse transcription polymerase chain reaction (RT-PCR). RT-PCR is characterized by high sensitivity and is used for relative quantitative assessment of gene expression on mRNA level. The initial phase while preparing to carry out the polymerase chain reaction is the primer design for specific gene. For successful realization of this process it is necessary to have some experience in work with information databases, nucleotide sequences and primer design. Also, it is necessary to find primers not only for specific genes but also for control gene. As a means of control, it is recommended to use the «housekeeping» gene or genes (constitutive gene), the expression of which is relatively constant regardless of external conditions. This fact will help to assess the change of expression of the target genes. This scope of work doesn't take a researcher a lot of time, but for a young explorer this search of nucleic acid sequences and primers can present a significant challenge. Wherewith, especially this phase of work is the initial stage of the initiation of molecular biological studies. All mistakes and inexperience could bring a great time andfinancial costs. In the present article a step-wise sequence of actions when searching for nucleotide sequences ofgenes and primer design to them are described, which may contribute to a general idea of the versatility, value and scope of bioinformatics approach. The description is made on a specific example using gene sequences of the constitutive protein tubulin of different plant objects. Described basic skills are quite enough for a young researcher to make a successful primer design, who has encountered the need for molecular studies for the first time.

Keywords

Authors

References