Restriction enzyme digestion of native and PCR-amplified DNA of an Acanthamoeba castellanii isolate

Noor Rain A¹, Rommel Q² , Sajiri S¹,Normaznah Y¹

1. Biotechnology Centre, Institute for Medical Research, Jalan Pahang 5038 Kuala Lumpur, Malaysia

2. Amboanga Medical School Foundation, Ateneo De Zamboanga, La Purisima ST, Zamboanga City 7000, Philippines

CITATION: Rain A, Rommel Q, Sajiri S, Normaznah Y. Restriction enzyme digestion of native and PCR-amplified DNA of an Acanthamoeba castellanii isolate. International Medical Research Journal. 1997 Jun;1(1):21–6. 

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ABSTRACT

This study attempted to verify the validity of using restriction fragment length polymorphism (RFLP) patterns of the genomic DNA and polymerase chain reaction (PCR) products of Acanthamoeba castellanii to assist in the identification and classification of the parasite. Both the genomic DNA and the PCR produces of the A. castellanii using a genus-specific set of primers A3 (TCCCCTAGCAGCITGTG) and A4 (GTTAAGGTCTCGTTCGTTA) were digested using 27 restriction endonucleases. The patterns of the digested products were analyzed by electrophoresis on agarose gel. The RFLP patterns of the genomic DNA showed different banding patterns with only enzyme Sma 1, which did not digest the DNA. The restriction endonuclease-digestion patterns of the PCR products showed that 4 restriction endonucleases; Alu I, BstN 1, Hinf I and Msp I digested the DNA. Our results showed that the RFLP patterns of the genomic DNA and the PCR products may be of use in identifying and classifying the A. castellani isolate chat we have.

KEYWORDS: Acanthamoeba castellanii, genomic DNA, restriction fragment length polymorphism, polymerase chain reaction