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Detection of variant of Hepatitis B virus and beta-globin through DNA technology
Author
Chia, Chee Poh
Supervisor
Tan, Ene Choo
Abstract
Two systems, i) the HBV precore genes, and ii) the human β-globin gene were used as models to develop simple, reliable and fast methods for the detection of point mutations. Three PCR-based methods were employed to detect the HBV precore stop codon mutant. They are RG-PCR, SSCP analysis and non-radioactive direct DNA cycle sequencing.
To test the feasibility of RG-PCR for detection of this HBV mutation, wild-type and mutant control templates which were generated through site directed mutagenesis were used. Amplification of these templates with a modified primer will create a new Dde I restriction site for the wild-type DNA but not the precore mutant DNA. Results showed that the mutant sample could be distinguished from the wild-type after restriction digestion with Dde I. RG-PCR was then used to detect the presence of the mutant viral DNA in patients' serum samples. Of forty eight serum samples which are HBsAg positive but HBeAg negative, 27 samples displayed the mutant pattern. Wild-type pattern was observed in only 8 samples, while another 7 samples showed a mixture of wild-type and mutant patterns. No result was obtained for the remaining 6 samples due to unsuccessful amplification.
Non-radioactive direct DNA cycle sequencing was subsequently employed for the detection and confirmation of the presence of the mutation in viral DNA in the serum samples. Sixteen samples tested with RG-PCR were sequenced. Nine samples had mutant sequence at nt 1896 and 3 samples had wild-type sequence. One sample showed both wild-type and mutant sequences. No bands were obtained for the remaining 3 samples. Other mutations were also found in the HBV precore/core gene. One was a frameshift mutation caused by the insertion of an A between nt 1836 and nt 1837. All the rest were base substitution mutations and were found in 13 different positions (nt 1846, 1848, 1899, 1916, 1933, 1938, 1975, 1977, 1984, 1992, 1993, 2002 and 2047).
A mini gel system was used for SSCP analysis. The optimal condition was first determined with four different sets of PCR products generated through site directed mutagenesis. Each set consists of a wild-type and a mutant sequence. Electrophoresis was carried out on gels of various acrylamide concentrations (6%, 10% and 18%) and different percentages of cross-linking (5%C, 3.3%C, 2.0%C, and 1.0%C). The best result for SSCP analysis was observed in gels with 18% acrylamide (2.0%C) and 5% glycerol.
SSCP analysis was carried out on samples which were previously characterized by RG-PCR. Two types of band patterns were observed for the wild-type samples, 8 for the precore stop codon mutant samples and 1 for the sample which has a mixture of wild-type and mutant. The many different patterns obtained showed that besides the stop codon mutation at 1896, there must be other base changes in the region. As results from DNA sequencing showed that this region of the HBV precore/core gene was highly polymorphic, SSCP analysis is therefore not suitable for the detection of this particular mutation.
SSCP analysis was also used to detect the presence of point mutations in the β-globin gene of 46 samples. Four pairs of primers and four different gel conditions (1X and 1.5X MDE gel), 18% acrylamide [2.0%C] with and without 5% glycerol} were tested. The best separation was seen on 18% acrylamide (2.0%C) gel with 5% glycerol. All but one of the mutations could be distinguished from the wild-type and from each other.
Unfortunately, the mutation which showed no mobility difference with the wild-type was the codon 41/42 (-TTCT) mutation, the most common in the local population. To detect this mutation by SSCP analysis, further optimization could be carried out with a shorter PCR fragment. In most cases, the heterozygous can be distinguished from the homozygous mutant. By using only four different sets of primers, 12 other mutations including the next 3 most common mutations nt -28 [A → G], codon 17 [A → T], and IVSI nt 5 [G → C] could be detected.
In conclusions, RG-PCR is useful for the detection of the HBV precore stop codon mutant although other mutations in the region will not be detected. SSCP is sensitive but it is not suitable for the detection of the particular HBV mutant as this region of the viral genome is highly polymorphic. On the other hand, SSCP is useful for screening mutations in β-globin gene when it is complemented by other methods. Non-radioactive direct DNA cycle sequencing can be used to detect and confirm mutations in the HBV genome.
Date Issued
1996
Call Number
QR201.H46 Chi
Date Submitted
1996