Plasmodium falciparum: Detection of Antifolate Resistance by Mutation-Specific Restriction Enzyme Digestion

The 4-aminoquinolines chloroquine (CQ) and amodiaquine (AM) were used to treat Gambian children with uncomplicated falciparum malaria in a randomized drug trial. Blood samples were taken immediately before treatment (day 0), and at day 7 and day 28 after treatment. Samples from those parasitologically positive at day 7 following treatment (‘early positives’) and those positive at day 28 but negative at day 7 (‘late positives’) have been studied by PCR followed by restriction enzyme digestion to determine the allelic status of the pfmdr 1 locus at the codon-86 position (asparagine or tyrosine), previously associated with resistance to CQ. A significantly higher prevalence of the tyr-86 allele was observed in samples taken immediately before treatment (day 0) in the early positives group when compared with the late positives group. This suggests the tyr-86 allele contributes to drug resistance in the early positives group. This association remained significant for both CQ and AM groups, implying a common genetic basis of resistance. Predominance of the allele at day 7 is consistent with a strong selection in the first week following treatment. In the late positives group, a significantly higher prevalence of the tyr-86 allele was observed in the samples at day 28 when compared with those at day 0, suggestive of selection during the period day 7 to day 28. Differences were observed in the extent of this selection in the CQ and AM groups. The samples were genotyped at 3 unlinked polymorphic loci. These analyses suggested that most parasites observed at day 7 were probably recrudescences whereas most of those at day 28 were reinfections.

Download Full-text

Isolation of Large-Terminal Sequences of BAC Inserts Based on Double-Restriction-Enzyme Digestion Followed by Anchored PCR

PCR Cloning Protocols ◽

10.1385/1-59259-177-9:337 ◽

2003 ◽

pp. 337-342

Author(s):

Zhong-Nan Yang ◽

T. Erik Mirkov

Keyword(s):

Restriction Enzyme ◽

Enzyme Digestion ◽

Restriction Enzyme Digestion ◽

Anchored Pcr

Download Full-text

Cytogenetic characterisation of Crassostrea gigas×C. angulata F1 hybrids: Restriction enzyme digestion chromosome banding and comparison of the aneuploidy levels of the two taxa and their hybrids

Aquaculture ◽

10.1016/j.aquaculture.2007.07.122 ◽

2007 ◽

Vol 272 ◽

pp. S284

Author(s):

A. Leitão ◽

R. Chaves ◽

F.M. Batista ◽

S. Santos ◽

H. Guedes-Pinto ◽

...

Keyword(s):

Restriction Enzyme ◽

Crassostrea Gigas ◽

Chromosome Banding ◽

Enzyme Digestion ◽

F1 Hybrids ◽

Restriction Enzyme Digestion

Download Full-text

Simple and robust screening of pooled yeast artificial chromosome libraries by the restriction enzyme digestion of polymerase chain reaction products

Genetic Analysis Biomolecular Engineering ◽

10.1016/1050-3862(94)90052-3 ◽

1994 ◽

Vol 11 (3) ◽

pp. 63-68 ◽

Cited By ~ 1

Author(s):

Xueqian Wang ◽

Jing Qian ◽

Minoru S.H. Ko

Keyword(s):

Polymerase Chain Reaction ◽

Restriction Enzyme ◽

Yeast Artificial Chromosome ◽

Artificial Chromosome ◽

Enzyme Digestion ◽

Restriction Enzyme Digestion ◽

Reaction Products ◽

Chain Reaction ◽

Polymerase Chain

Download Full-text

Isolation of large terminal sequences of BAC inserts based on double-restriction-enzyme digestion followed by anchored PCR

Genome ◽

10.1139/g99-120 ◽

2000 ◽

Vol 43 (2) ◽

pp. 412-415 ◽

Cited By ~ 4

Author(s):

Zhong-Nan Yang ◽

T Erik Mirkov

Keyword(s):

Restriction Enzyme ◽

Genomic Dna ◽

Enzyme Digestion ◽

Restriction Enzyme Digestion ◽

Artificial Chromosomes ◽

Bac Clones ◽

Anchored Pcr ◽

Pcr Products ◽

Left And Right ◽

The Right

Isolation of the terminal portions of genomic DNA cloned in bacterial artificial chromosomes (BACs) is an important step in map-based cloning, and several methods have been developed. Here, we present a new method based on double-restriction-enzyme digestion followed by anchored PCR. BAC DNA was digested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini. After dephosphorylation, these digestions were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector developed in this work that is derived from pBluescript SK(+). PCR products representing the left- and right-terminal sequences of BAC inserts were obtained using a primer complementary to pMSK and a primer complementary to sequences in either the left arm or the right arm of the BAC vector pBeloBAC11. We have tested this method with 15 different BAC clones, and PCR products representing both the left- and right-terminal sequences have been obtained from all 15 BAC clones. This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used. With some modifications, it can also be used for isolating the terminal portions of genomic DNA cloned in yeast artificial chromosomes and P1-derived artificial chromosomes. Key words: BAC, anchored PCR, terminal sequence isolation, chromosome walk.

Download Full-text