Inexpensive, Rapid and Convenient PCR-minigel SSCP
Protocol for Polymorphisms and Mutations Analyses of
LDL Receptor Gene
KLAI-UPSORN S. PONGRAPEEPORN, Ph.D.*,
PIKUN THEPSURIYANON, M.Sc.*****,
WATTANA LEOWATTANA, M.D.**,
SOMPONG ONG-AJYOOTH, M.Sc.*,
SUDCHAREE KIARTIVICH, M.Sc. **,
PREYANUJ YAMWONG, M.D.***
LUMPOON KASEMSUK, B.Sc.*,
KWANDOA KERDSAENG,B.Sc.*,
WILAIRAT NUCHPRAMOOL, M.D.****,
SIV ADEE LAUNGSUW AN, M.Sc. **,
ANCHALEE AMORNRATTANA, M.Sc.****,
Affiliation : * Department of Biochemistry,
** Department of Clinical Pathology,
*** Department of Preventive and Social Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University,
Bangkok, 10700,
**** Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Bangkok, 10700,
Thailand.
***** Amersham Pharmacia Biotech, South East Asia.
AbstractHypercholesterolemia has been recognized as a major risk factor of atherosclerosis and
coronary artery disease. The elevation in plasma low density lipoprotein (LDL) cholesterol is fre-
quently due to genetic alteration at the genetic locus specifying the LDL receptors, leading to
defective catabolism of LDL. In order to facilitate the molecular diagnosis of LDL receptor disorder,
single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR)
amplified genomic DNA fragments has become a simple and sensitive screening method for iden-
tification of DNA polymorphisms and mutations in LDL receptor gene prior to DNA sequencing.
In addition, SSCP patterns can be detected by silver staining to avoid hazardous radioactive material
or other costly nonradioactive detection techniques. However, the original SSCP protocol is
generally large-formatted, which is both time and reagents consuming as well as cumbersome.
Minigel SSCP protocols have thus been devised but they involve, although commercially
. available, costly precast gels. We describe here a nonradioactive PCR-minigel SSCP protocol which
is sensitive, inexpensive, rapid, reproducible and manually convenient. The results in this study
demonstrate that minigel-SSCP (gel size: 10 em x 7.3 em x 0.075 em) can detect conformation
polymorphisms in PeR-fragments with a comparative sensitivity to large gel SSCP (gel size: 30
em x 40 em x 0.04 em) as exemplified by the SSCP analyses of exon 13 of the LDL receptor gene.
For minigel SSCP, the reagents for gel components and silver staining are reduced approximately
9 times and 10 times, respectively. For electrophoresis, electrical power is also reduced 10 times.
This improved technique can become routinely used for molecular diagnosis of LDL receptor
defect as well as for other genetic disorders.
Keywords : Hypercholesterolemia, LDL Receptor Gene, Polymorphism, Mutation, PCR, Minigel
SSCP
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