Primer design and restriction site analysis targeting POU domain class 1 transcription factor 1 gene of Friesian Holstein in silico

This study was conducted to design primer set and to map restriction site targeting synonymous mutation c.297A>G of the POU domain class 1 transcription factor 1 gene (POU1F1) of Friesian Holstein in silico. Primer set was designed to target exon 3 of the POU1F1 based on nucleotide sequence at GenBank database with accession number NC_037328.1 using primer3. Simulations of amplification targeting POU1F1 fragment and restriction fragment length polymorphism (RFLP) were conducted in silico using serial cloner version 2.6 and NEBcutter version 3.0, respectively. This study used a primer pair (5′-CCT TTT AGA ACT GAG ACT GGC TG-3′ and 5′-CCC ACA GCT GTT AAC AAG CA-3′) that produce 356 bp of estimated product size. Moreover, a synonymous mutation, c.297A>G of the POU1F1, could be detected using BsII restriction enzyme in silico. The BsII did not have restriction site for AA genotype. On the other hand, it could cut the PCR product size into two fragments (167 and 189 bp) for GG genotype. It can be concluded that in silico analysis successfully amplified target region using primer designed in this study and RFLP simulation using BsII could detect synonymous mutation c.297A>G of the POU1F1. Further in vitro study should be conducted to identify c.297A>G in Friesian Holstein population.


Introduction
The POU domain class 1 transcription factor 1 (POU1F1) gene in Friesian Holstein (FH) dairy cattle is involved in mammary gland development that can control quality and production traits of milk.It is a transcription factor that controls the promoters of GH, PRL and TSH [1].POU1F1 gene consists of five introns and six exons, the gene is mainly synthesized in the anterior pituitary [2].The POU1F1 gene affects pituitary and mammary gland development, expression of milk protein, and production of milk and able to produce proteins that are thought to control milk production and quality traits [3].POU1F1 also has functions as an activator of the pituitary gland that controls growth hormone in several mammalian species [4].The POU1F1 gene can be utilized in selection using the concept of marker assisted selection (MAS) that uses polymorphic locus information as an aid to select superior dairy cows.
Bioinformatics is an advance science in computational biology and primarily a knowledge discipline that handles information of genetics for research, development, or computational applications and approaches to obtain, deposit, visualize, and construe biological data [5].Designing of primer by using computational biology is crucial tools for amplification of regions of DNA target.The primer serves as a delimiter of the target fragment of DNA to be produced [6].Furthermore, restriction site analysis of DNA variations could be conducted by computer simulation methodology which is well known as in silico study to predict, hypothesize, and provide new discoveries in research [7].
Variations within a gene can be analysed by predicting the restriction site of specific regions in silico.Potential polymorphisms within the POU1F1 gene among individual Friesian Holstein cattle enable to be unravelled by using restriction endonuclease enzymes that digest DNA fragments into different sized or length.This technique is widely used in genetic engineering studies and known as restriction fragment length polymorphisms (RFLP).Previous studies reported that some single nucleotide polymorphisms (SNPs) have been found within both intronic and exonic regions of the POU1F1 in vitro [8][9].In this study, designing of primer and restriction site analysis of POU1F1 gene within the genomic landscape of Friesian Holstein cattle was employed to find out novel and potential SNP for uncovering genetic insights that could shape the future of dairy breeding and production.

Materials and methods
This study is computational work using biological data in silico.Therefore, it did not require ethical clearance since there were no experimental animals involved.

Identification of the POU1F1 polymorphism
Identification of the POU1F1 polymorphism was conducted by exploring database of the POU1F1 sequence at the GenBank website (https://www.ncbi.nlm.nih.gov/).In addition, Ensembl genome browser (https://www.ensembl.org/)was also used to find out candidate mutations which are able to be recognized by restriction endonuclease enzymes.Point of mutation selected in this study was c.297A>G of the POU1F1.

Primar design targeting POU1F1 polymorphism and PCR in silico
Primer design was prepared using reference sequences obtained from the NCBI site with accession number NC_037328.1.Primer design is the first step in obtaining the best primer sequences for DNA amplification so that it can be used for in vitro analysis of samples using the Polymerase Chain Reaction (PCR) technique.In addition, PCR is also used to validate the results of gene expression assays and is able to produce large number of data [10][11].Primer pair was designed using primer3 web version 4.1.0targeting exon 3 of the POU1F1 gene of FH cattle.The primers used in this study were novel primers prepared by a series of primer design stages, with parameters in the form of several primary properties which include product length, base length nucleotide, percentage of G and C (%GC), melting point (Tm), and secondary properties namely hairpin, self-dimer, and heterodimer [12].Moreover, the primers (Table 1) were simulated using the Serial Cloner version 2.6 application to test whether the primers would attach or not to the genomic sequence.Reverse 5'-ccc aca gct gtt aac aag ca-3'

Restriction enzyme selection and PCR-RFLP in silico
The restriction enzyme used was selected in according to the target mutation point.Selection and simulation of restriction enzymes were carried out using the Nebcutter web version 3.0.Restriction endonuclease enzyme which is able to recognize c.297A>G of the POU1F1 was BsII.

Identification of the POU1F1 polymorphism
Identification of POU1F1 polymorphism can be explored using Genbank database and Ensembl genome browser.There were seven polymorphisms identified in the exon 3 of the POU1F1 using these platforms (Table 2).Of these, the c.297A>G of the POU1F1 can be detected by BsII restriction enzyme in silico.
Previous studies reported that the POU1F1 variations have been revealed to be significantly related to economically important quantitative traits such as milk production, reproductive traits such as litter size, and body weight in various cattle breeds [8][9].

Primar design targeting POU1F1 polymorphism and PCR in silico
Primer designed in this study were forward primer 5'-CCT TTT AGA ACT GAG ACT GGC TG-3' and reverse primer 5'-CCC ACA GCT GTT AAC AAG CA-3'.The length of forward primer was 23 nucleotides and reverse primer was 20 nucleotides.The primers designed in this study were carried out in silico and successfully amplified target region sized 356 bp (Figure 1).Success PCR depends on the way unique oligonucleotide primers recognize target sequence efficiently.Primers used in PCR technique are usually 18-35 bases in length and serve to hybridize with the DNA template and determine amplified region, and also the place of attachment of DNA polymerase to amplify the target fragment of the gene [13][14].Moreover, melting temperature (Tm) of the forward and reverse primers were respectively 55.3 and 55.7°C, which are good for reaction due to small different between them.These temperatures are affected by length of primer [15].

Figure 1.
The PCR simulation in silico using primer pair designed in this study PCR is a technique used to identify species derivatives molecularly at the deoxyribonucleic acid (DNA) level [16].PCR, a scientific term and molecular biology technique, can copy particular DNA segment by using two short oligodeoxynucleotide sequences known as primers through a repetitive thermal reaction facilitated by a DNA polymerase enzyme [17].The advantages of the PCR technique include high specificity and high sensitivity when compared to other techniques [18].Then, PCR is also able to identify a large number of samples with little risk of contaminants [19].The success of the PCR reaction was simulated using the Serial Cloner application which is a bioinformatic tool used to organize a cloning project, do DNA alignment and amino acid sequences, and simulate sub-cloning events.The DNA fragments in the form of PCR products located at certain places will be copied by DNA polymerase in the application to avoid errors so that it can be continued with the in vitro amplification.

Restriction enzyme selection and PCR-RFLP in silico
The c.297A>G SNP of the POU1F1 is a synonymous mutation that do not lead to changes in the activity of the product encoded by the gene [20].BslI restriction enzyme, a type II restriction endonuclease purified from Bacillus sp.[21], was selected to detect c.297A>G SNP.The BslI enzyme successfully cut into DNA fragments whereas restriction enzymes producing undetectable or tiny size of DNA fragments and also showed no variability in profiles of digestion were excluded [22].The BslI has restriction site and recognizes the nucleotide sequence of 5′-CCNNNNN ↓ NNGG-3′ within the PCR product.The BslI did not have restriction site for AA genotype.On the other hand, it could cut the PCR product size into two fragments (167 and 189 bp) for GG genotype (Figure 2).

Figure 2 .
Figure 2. The PCR-RFLP simulation in silico 4. Conclusion A synonymous mutation of the POU1F1, c.297A>G, has been successfully amplified using primer designed in this study and RFLP simulation using BsII could detect variations of c.297A>G in silico.Further in vitro study should be applied to identify c.297A>G in Friesian Holstein population.

Table 1 .
Primer designed targeting POU1F1 exon 3 of the FH cattle.