Recombinant chimeric sequences originating from a mix of the sequences of two different alleles are frequently found after amplification and cloning in E.coli of the exon 2 of the Major Histocompatibility Complex (MHC) DRB genes. Several authors suggested that the recombinant molecules are due to in vitro recombination during PCR, nevertheless a clear experimental demonstration of this hypothesis is missing. In order to understand the mechanism producing the chimeric sequences, we set up a simple experiment based on the different restriction patterns of parental and recombinant sequences. Our data demonstrate that in the analysed case most of the recombinant variants are not produced by in vitro recombination during PCR, but are the result of the mismatch repair of heteroduplex molecules during cloning in E. coli. The high mutation rate in the -helix region of DRB expressed genes, both after cloning in E.coli and after the germ-line differentiation process of vertebrates, suggests that the observed mutations are the result of similar gene conversion processes, probably favoured by Chi-dependent micro-recombination events
Recombinant DRB sequences produced by mismatch repair of heteroduplexes during cloning in Escherichia coli / M. Longeri, M. Zanotti, G. Damiani. - In: EUROPEAN JOURNAL OF IMMUNOGENETICS. - ISSN 0960-7420. - 29:6(2002), pp. 517-523.
Recombinant DRB sequences produced by mismatch repair of heteroduplexes during cloning in Escherichia coli
M. LongeriPrimo
;M. ZanottiSecondo
;
2002
Abstract
Recombinant chimeric sequences originating from a mix of the sequences of two different alleles are frequently found after amplification and cloning in E.coli of the exon 2 of the Major Histocompatibility Complex (MHC) DRB genes. Several authors suggested that the recombinant molecules are due to in vitro recombination during PCR, nevertheless a clear experimental demonstration of this hypothesis is missing. In order to understand the mechanism producing the chimeric sequences, we set up a simple experiment based on the different restriction patterns of parental and recombinant sequences. Our data demonstrate that in the analysed case most of the recombinant variants are not produced by in vitro recombination during PCR, but are the result of the mismatch repair of heteroduplex molecules during cloning in E. coli. The high mutation rate in the -helix region of DRB expressed genes, both after cloning in E.coli and after the germ-line differentiation process of vertebrates, suggests that the observed mutations are the result of similar gene conversion processes, probably favoured by Chi-dependent micro-recombination eventsFile | Dimensione | Formato | |
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