Multiplexed genetic analysis using an expanded genetic alphabet.
BACKGROUND
All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening.


METHODS
MultiCode PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in approximately 3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns.


RESULTS
In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls.


CONCLUSIONS
The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting.
Johnson
Scott C
SC
Eragen Biosciences, Inc., Madison, WI 53717, USA.
Marshall
David J
DJ
Harms
Gerda
G
Miller
Christie M
CM
Sherrill
Christopher B
CB
Beaty
Edward L
EL
Lederer
Scott A
SA
Roesch
Eric B
EB
Madsen
Gary
G
Hoffman
Gary L
GL
Laessig
Ronald H
RH
Kopish
Greg J
GJ
Baker
Mei Wang
MW
Benner
Steven A
SA
Farrell
Philip M
PM
Prudent
James R
JR
D001322:Autoanalysis; D003550:Cystic Fibrosis / Q000175:diagnosis; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005838:Genotype; D006801:Humans; D007231:Infant, Newborn; D009154:Mutation; D015997:Neonatal Screening / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012189:Retrospective Studies; D012371:Robotics; D012984:Software
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator