The use of RAPDs for the analysis of parasites.
There is a lack of sequence information concerning polymorphic loci in parasite genomes. Thus, the use of arbitrary PCR primers under low temperature annealing conditions to generate random amplified polymorphic DNAs (RAPDs) represents an important approach to the study of the structure of parasite populations, their genetic variation as well as improved diagnosis of the diseases they cause. Following the examination of all variables and their effect on the reproducibility of the reaction, we have established a protocol for the analysis of RAPDs that involves amplification at two separate DNA concentrations followed by polyacrylamide gel electrophoresis and silver staining. We find the technique to be sensitive, reproducible, simple and relatively cheap. It has already provided insight into the genetic variation in populations of schistosomes and trypanosomes and is being used to study various other endemic infections. We also use specific primers under low stringency conditions in situations where the objective of the amplification is the detection of a particular sequence and where normal high stringency conditions give a positive/negative answer such as sex determination or diagnosis of blood born infections. Under low stringency conditions, specific amplification products persist but products of low stringency priming are also apparent and serve as a perfect internal control for negative samples.
Laboratório de Bioquímica e Biologia Molecular, Centro de Pesquisas René Rachou, Belo Horizonte, Minas Gerais, Brazil.
A J
AJ
Simpson
E
E
Dias Neto
M
M
Steindel
O L
OL
Caballero
L K
LK
Passos
S D
SD
Pena
D000818:Animals; D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods* / Q000706:statistics & numerical data; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D005260:Female; D005819:Genetic Markers; D008297:Male; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D012550:Schistosoma mansoni / Q000235:genetics*; D012732:Sex Determination Analysis / Q000379:methods; D014349:Trypanosoma cruzi / Q000235:genetics*
D017931:DNA Primers; D016054:DNA, Protozoan; D005819:Genetic Markers