THE USE OF AN INTERNAL STANDARD MOLECULE FOR DETERMINING MUTATION FREQUENCY IN RESTRICTION SITE MUTATION METHOD : SINIRLI BÖLGE MUTASYON YÖNTEMİ İLE MUTASYON FREKANSININ BELİRLENMESİ İÇİN İÇ STANDART

In order to overcome some problems associated with the effects of variables on in vitro amplification of specific DNA segments using polymerase chain reaction (PCR), an internal standard technique was included to restriction site mutation (RSM) method. The internal standard was designed with same nucleotide sequences as the target sequence but with a deletion so that differences in size could be used to distinguish the PCR products of the target and internal standard. The application of the internal standard usage in the RSM method was performed on the rat ras gene. N-methyl-N-nitrosourea (MNU) induced mutations were detected at frequencies 10at the H-ras gene in exon 1 at Hind III restriction enzyme recognition sequence. The use of internal standard successfully allowed the calculation of mutation frequency.