Quantitative Restriction Fragment Length Polymorph...
Here we present an assay for quantitation of a particular gene allele in DNA mixtures by means of restriction fragment length polymorphism（RFLP）in combination with polymerase chain reaction（PCR）.We applied the quantitative RFLP principle for estimation of the relative amount of diphtheria toxin gene CRM197 allele in Corynebacterium diphtheriae culture DNA samples.The procedure is based on PCRmediated generation of an artificial AluI restriction site specifically with the CRM197 DNA template.After AluI digestion of the PCR product and polyacrylamide gel electrophoresis of the restriction fragments，the percentage of CRM197 template in the initial DNA sample was determined by scanning a gel negative.The method was shown to give a linear response when applied to template mixtures containing different amounts of CRM197 reference template.For samples where non-CRM197 DNA was detected by AluI RFLP，we designed a further allele-specific PCR assay to determine whether the non-CRM197 template portion was the wild-type toxin gene allele.
In the biopharmaceutical industry，it is important to evaluate the genetic stability of a recombinant protein producing strain because of a concern that mutational alterations may occur in critical DNA sequences during cultivation.This is particularly the case for cultures of Corynebacterium diphtheriae which produce the inactive mutant form of diphtheria toxin protein，CRM197.
Diphtheria toxin protein，secreted by pathogenic strains of C.diphtheriae，is the causative agent for diphtheria in humans［1］.This 58-kDa protein is able to kill eukaryotic cells by blocking cellular protein syn thesis［2-5］.Toxin protein is encoded by the tox gene of Corynebacteriophage β integrated into the chromosome of lysogenic Corynebacterium［6，7］.
Chiron Corporation，4560 Horton Street，Emeryville，California 94608-2916
Received January 14，1998(https://www.daowen.com)
此文发表于1998年“Anal.Biochem.”260：24-29.
A number of tox mutants which produce altered toxin proteins，designated CRMs（serologically cross-reacting materials），have been isolated by mutagenic treatment of Corynebacteriophageβ with nitrosoguanidine.CRMs are either completely non-toxic or of greatly reduced toxicity，yet still cross-react with diphtheria antitoxin［8-10］.From the point of view of active immunization against diphtheria，the most interesting of the CRMs that have been isolated is CRM197.Due to a G→A transition in the tox gene leading to a GLY→GLU substitution at position 52，CRM197 protein is enzymatically inactive，but appears to be serologically indistinguishable from wild-type toxin［2，8，9，11］.In addition to its use for immunization against diphtheria，CRM197 protein is successfully used as an adjuvant protein carrier for carbohydrate antigens［1，12］.
Considering the possibility of CRM197 reversion to wild-type during cultivation of bacteria，it is very important to be able to monitor the genetic stability of CRM197 mutation during the process of protein production.The purpose of the present study was to develop a simple assay to quantitate the relative amount of the CRM197 mutant allele in Corynebacterium genomic DNA samples，and to distinguish between CRM197 and wild-type tox gene alleles.
For quantitation of the CRM197 mutant allele，a portion of the toxin gene was polymerase chain reaction（PCR）[2]-amplified with primers generating an artificial Alul restriction site specifically with the CRM197 DNA template.The resulting PCR product was digested with AluI，and restriction fragments were separated in a polyacrylamide gel.The percentage of CRM197 template in the initial DNA sample was determined by scanning the gel negative.Since the quantitative RFLP assay did not allow to detect the wildtype tox gene allele specifically，a complementary allele-specific PCR assay was designed to determine the presence of the wild-type allele.
