An improved polymerase chain reaction (PCR) protocol for unambigous detection of growth hormone gene deletions.
hGH-1 gene deletions are detected by simultaneous PCR amplification along the two homologous DNA sequences flanking the hGH-1 gene on both sides and are differentiated by SmaI restriction enzyme digestion. We have observed that among the SmaI digested PCR products from normal homozygous subjects, from those heterozygous for the 7.6 kb deletion and from those heterozygous for a 6.7 kb deletion, along with the expected fragments there is an unexpected 1470 bp fragment. This fragment arises from the co-amplification of a third homologous sequence located downstream from the hGH-1 gene and it confuses differentiation between normal homozygous and heterozygous for 7.6 kb subjects from the 6.7 kb heterozygous subjects. To overcome this problem we have improved PCR conditions using a different reverse primer. These changes avoid the interaction of the primers with the third homologous sequence located downstream from the hGH-1 gene and prevent the appearance of this additional band that complicates the interpretation of the results. We conclude that the new reverse primer sequence avoids the amplification of the downstream hGH-1 gene sequence and the production of the 1474 bp band after SmaI endonuclease enzyme digestion and makes it possible to differentiate homozygous normal subjects and those who are heterozygous for a 7.6 kb deletion from those who are heterozygous for a 6.7 kb deletion.
Mone
C M
CM
Institute of Endocrinology, Second University of Naples, Italy.
Nigro
V
V
Rotondi
M
M
Del Buono
A
A
Mazziotti
G
G
Riondino
M
M
Sinisi
A M
AM
Ghizzoni
L
L
Phillips
J A
JA
Bellastella
A
A
Carella
C
C
D004996:Ethidium; D005456:Fluorescent Dyes; D017353:Gene Deletion; D006580:Genetic Carrier Screening; D006720:Homozygote; D019382:Human Growth Hormone / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D005456:Fluorescent Dyes; D019382:Human Growth Hormone; D004996:Ethidium