Use of multiple displacement amplification to amplify genomic DNA before sequencing of the alpha and beta haemoglobin genes.
OBJECTIVE
To evaluate the technique of multiple displacement amplification (MDA) for whole genome amplification from small volume blood samples before sequencing in a clinical test to identify haemoglobin gene mutations.


METHODS
Phage phi29 DNA polymerase was used to perform MDA, starting with either 1 micro l of blood or 1 ng of previously isolated blood DNA from 23 patients. The amplified products were then evaluated using a clinical test that involves sequencing the haemoglobin genes to detect mutations. The results were compared with the current clinical test method that uses genomic DNA isolated using column based technology.


RESULTS
The MDA technique produced large quantities (theoretically approximately 2 mg) of DNA. The amplification procedure was extremely easy and took about four hours (less than one hour of hands on technician time and three hours for amplification). When MDA products were used in the same clinical test protocol as genomic DNA isolated using column technology, there was 100% concordance for detection of a variety of point mutations in the alpha1, alpha2, and beta globin genes.


CONCLUSIONS
The MDA technique is useful for overcoming the problem of insufficient genomic DNA in clinical specimens requiring haemoglobin gene sequencing and could be useful for other clinical applications.
Mai
M
M
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester 55905, USA.
Hoyer
J D
JD
McClure
R F
RF
D001483:Base Sequence; D004587:Electrophoresis, Agar Gel; D015894:Genome, Human; D005914:Globins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA
D005914:Globins