VISUALIZAÇÃO DE RESUMO

C. Ciências Biológicas - 8. Genética - 4. Genética Molecular

A SIMPLE PERL SCRIPT TO FIND PARTIALLY CONSERVED RESTRICTION SITES IN TEMPLATE DNAS

André Luis Rodrigues ¹

Luismar Marques Porto ¹

(1. Departamento de Engenharia Química e Engenharia de Alimentos (EQA/CTC), UFSC)

INTRODUÇÃO:

Primer design for amplification of DNA sequences by polymerase chain reaction (PCR) often involves the use of degenerated oligos. In the case of directional cloning, double digestion reactions are employed to ensure correct insertion of the template DNA into the vector. If the template DNA does not contain the restriction sites (RS) to be used, they need to be included in the primers for subsequent double digestion of the amplicon. Insertion of mutations in primers may avoid proper annealing during PCR. Thus, these insertions should be maximally reduced. A simple Perl script was developed in order to localize partial conserved RS in template DNA sequences. The script seeks for an input RS sequence in a template DNA sequence and its variations in one and two positions. The returned sequences, as well as their positions are saved into a text file. The developed script is potentially useful in the design of degenerated primers for PCR cloning.

METODOLOGIA:

An available freeware (ActivePerl 5.6.1.638 for MS-Windows) was used to write the script. Enzymatic digestion was simulated using a common text editor. Basically, the developed script is divided into four subroutines. The first extracts the template DNA sequence from an input file and converts it to a single string. Two other subroutines generate variations in one and two positions of an input RS sequence. Finally, the last subroutine seeks for the input RS and its variations over the template DNA sequence and returns the searched sequences and their respective positions. As a test case, the template DNA sequence used was: acgaaaggcagcatcccgattccaaggcgcgagtgccgtaggttttcgccgcccatgcccgtccgccgttgccgcgcggcgggcgtgagttgactagtcaaaaggacattcgcgATG. The input XhoI RS was ctcgag.

RESULTADOS:

The input of the RS into the script resulted in six partially conserved RS, one RS with one mutation and five RS with two mutations. Among the RS identified, cgcgat was selected due to its position. The sequence cgcgat was the only one that allowed the promoter of the template DNA sequence to be excluded. An output example is shown below, where the a.dna file contains the input sequence.

Enter the name of the template DNA file: a.dna

Enter the RS to be searched: ctcgag

Intact sites: No site found

Sites with one mutation: Found cgcgag at 30

Sites with two mutations: Found ttcgcg at 111, Found cccgat at 17

Found cgtgag at 86, Found cgcgcg at 75, Found cgcgat at 113

The sequence cgcgat in the template DNA was substituted by the sequence ctcgag to simulate enzymatic digestion. The simulation of enzymatic digestion and ligation of the mutated template DNA and the vector generated a DNA fragment which is in frame with the promoter of the vector (1). Bold letters denote the inserted RS, ATG is the start codon of the vector, as seen below:

accATG.gat.ccg.agc.tcg.agg.aag.cat.tct.tcc.gat.atc (1)

M D P S S R K H S S D I (2)

M K H S S D I (3)

The codified protein (2) contains five amino acids more than the original protein (3). The first amino acid of the original protein, Methionine (M), is substituted by one Arginine (R).

CONCLUSÕES:

A simple Perl script was successfully developed to seek and identify partially conserved RS. The script might be improved to simulate and evaluate enzymatic digestions of partially conserved RS in template DNAs. Therefore, computational simulation of directional cloning could be less time-consuming.

Instituição de fomento: Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq

Palavras-chave: primer design; partially conserved restriction sites; cloning.

Anais da 58ª Reunião Anual da SBPC - Florianópolis, SC - Julho/2006