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Detection of mutations and polymorphisms in the human beta-globin gene and hepatitis B virus pre-core region by Denaturing Gradient Gel Electrophoresis (DGGE)
Author
Phang, Beng Hooi
Supervisor
Tan, Ene Choo
Abstract
DNA carries biological information in a form that must be precisely copied and transmitted to all progeny cells. Occasionally, changes in the sequence of a gene may occur during the replication process. These "genetic mistakes" are known as mutations. Mutations can be identified by physical, chemical or enzymatic means. In this study, mutations in the human B-globin gene and HBV pre-core gene were used to evaluate the sensitively of Denaturing Gradient Gel Electrophoresis (DGGE). DNA sequencing was then performed to verify and confirm the unknown mutations in the human B-globin gene as well as the HBV pre-core gene.
Different regions of the B-globin gene were obtained via Polymerase Chain Reaction (PCR) amplification and the products were subjected to DGGE. Modifications to standard protocols were made to establish better electrophoretic and gel conditions to improve the resolution of bands as well as to increase the sensitivity of the system. All the different mutations were detected and the band pattern for each mutation was different. The presence of polymorphisms was also detected by DGGE as different band patterns. Results were found to be highly reliable. Changes in samples with unknown B-globin mutations were detected by DGGE and were subsequently verified by sequencing. DGGE was also used in antenatal diagnosis. Results from DGGE analysis of antenatal samples were found to be highly reliable.
For the detection of HBV pre-core mutant (mutation at nt 1896) using DGGE, results were not promising. The region of interest for DGGE analysis was obtained via PCR. Bands at different positions were observed. The pre-core region of these samples were sequenced and changes in other nucleotides were found. As a result, band positions that were obtained from control samples could not be used as references to identify the presence of nt 1896 mutation in serum samples.
In conclusion, DGGE has proven to be a highly sensitive method in the detection of mutations with an efficiency approaching 100%. It is suitable for the detection of single substitution mutation which occurs in any position along a DNA fragment which is highly conserved. Its application in prenatal diagnosis shows promise in future as a screening method. However, it might not be useful if the region is highly polymorphic.
Different regions of the B-globin gene were obtained via Polymerase Chain Reaction (PCR) amplification and the products were subjected to DGGE. Modifications to standard protocols were made to establish better electrophoretic and gel conditions to improve the resolution of bands as well as to increase the sensitivity of the system. All the different mutations were detected and the band pattern for each mutation was different. The presence of polymorphisms was also detected by DGGE as different band patterns. Results were found to be highly reliable. Changes in samples with unknown B-globin mutations were detected by DGGE and were subsequently verified by sequencing. DGGE was also used in antenatal diagnosis. Results from DGGE analysis of antenatal samples were found to be highly reliable.
For the detection of HBV pre-core mutant (mutation at nt 1896) using DGGE, results were not promising. The region of interest for DGGE analysis was obtained via PCR. Bands at different positions were observed. The pre-core region of these samples were sequenced and changes in other nucleotides were found. As a result, band positions that were obtained from control samples could not be used as references to identify the presence of nt 1896 mutation in serum samples.
In conclusion, DGGE has proven to be a highly sensitive method in the detection of mutations with an efficiency approaching 100%. It is suitable for the detection of single substitution mutation which occurs in any position along a DNA fragment which is highly conserved. Its application in prenatal diagnosis shows promise in future as a screening method. However, it might not be useful if the region is highly polymorphic.
Date Issued
1997
Call Number
QH461 Pha
Date Submitted
1997