Multiplex Loss of Heterozygosity Analysis by Using Single or Very Few Cells

Cui, Xiangfeng; Feiner, Helen; Li, Honghua

doi:10.1016/s1525-1578(10)60699-x

Cited by 4 publications

(4 citation statements)

References 19 publications

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“…Our group has already developed a highly efficient multiplex PCR protocol that can be used to amplify a large number of loci that could be resolved either by conventional PAGE or by denaturing gradient gel electrophoresis (Lin et al 1996;Cui and Li 1998;Cui et al 2000). Very recently, we showed that 10 independent sequences may be amplified simultaneously from single diploid cells (Cui et al 2002). In the present study, we used a subgrouping strategy in the second round of amplification and successfully amplified 32 marker sequences from single sperm, compared with 5 or 6 in most studies with large amounts of DNA.…”

Section: Discussionmentioning

confidence: 99%

Direct Detection of Insertion/Deletion Polymorphisms in an Autosomal Region by Analyzing High-Density Markers in Individual Spermatozoa

Pramanik¹,

Li²

2002

The American Journal of Human Genetics

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Direct polymerase chain reaction (PCR) detection of insertion/deletion (indel) polymorphisms requires sample homozygosity. For the indel polymorphisms that have the deletion allele with a relatively low frequency in the autosomal regions, direct PCR detection becomes difficult or impossible. The present study is, to our knowledge, the first designed to directly detect indel polymorphisms in a human autosomal region (i.e., the immunoglobulin V(H) region), through use of single haploid sperm cells as subjects. Unique marker sequences (n=32), spaced at approximately 5-kb intervals, were selected near the 3' end of the V(H) region. A two-round multiplex PCR protocol was used to amplify these sequences from single sperm samples from nine unrelated healthy donors. The parental haplotypes of the donors were determined by examining the presence or absence of these markers. Seven clustered markers in 6 of the 18 haplotypes were missing and likely represented a 35-40-kb indel polymorphism. The genotypes of the donors, with respect to this polymorphism, perfectly matched the expectation under Hardy-Weinberg equilibrium. Three V(H) gene segments, of which two are functional, are affected by this polymorphism. According to these results, >10% of individuals in the human population may not have these gene segments in their genome, and approximately 44% may have only one copy of these gene segments. The biological impact of this polymorphism would be very interesting to study. The approach used in the present study could be applied to understand the physical structure and diversity of all other autosomal regions.

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Section: Discussionmentioning

confidence: 99%

Direct Detection of Insertion/Deletion Polymorphisms in an Autosomal Region by Analyzing High-Density Markers in Individual Spermatozoa

Pramanik¹,

Li²

2002

The American Journal of Human Genetics

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“…However, low amounts and often poor quality of DNA isolated from such material represent a serious obstacle to large-scale mutational studies. 4,5 Human tumors contain a high proportion of C to T transition mutations. 6 Human uracil-DNA glycosylase (UNG) is a major DNA glycosylase that counteracts accumulation of such mutations in the genome.…”

Section: (J Mol Diagn 2005 7:36 -39)mentioning

confidence: 99%

Low Copy Number DNA Template Can Render Polymerase Chain Reaction Error Prone in a Sequence-Dependent Manner

Akbari

Hansen

Halgunset

et al. 2005

The Journal of Molecular Diagnostics

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Paraffin-embedded tissue is an important source of material for molecular pathology and genetic investigations. We used DNA isolated from microdissected formalin-fixed, paraffin-embedded gastric tumors for mutation analysis of a region of the human gene for uracil-DNA glycosylase (UNG), encoding the UNG catalytic domain, and detected apparent base substitutions which, after further investigation, proved to be polymerase chain reaction (PCR) artifacts. We demonstrate that low DNA template input in PCR can generate false mutations, mainly guanine to adenine transitions, in a sequence-dependent manner. One such mutation is identical to a mutation previously reported in the UNG gene in human glioma. This phenomenon was not caused by microheterogeneity in the sample material because the same artifact was seen after amplification of a homogenous, diluted plasmid. We did not observe genuine mutations in the UNG gene in 16 samples. Our results demonstrate that caution should be taken when interpreting data from PCR-based analysis of somatic mutations using low amounts of template DNA, and that methods used to enrich putative subpopulations of mutant molecules in a sample material could, in essence, be a further amplification of sequence-dependent PCR-generated artifacts.

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“…PCR primers are designed based on the selected sequences, so that amplification with these primers generates PCR products of different lengths and can be resolved by regular gel electrophoresis. Experimentally, the marker sequences are amplified with a two‐round multiplex protocol developed in our laboratory (112–114). If the number of marker sequences is large, the sequences may be amplified separately in different groups in the second round to ensure the resulting fragments are easily resolved.…”

Section: New Tools For Understanding Gene Segment Organization In Thementioning

confidence: 99%

Genetic diversity of the human immunoglobulin heavy chain V_Hregion

Cui

Pramanik

et al. 2002

Immunological Reviews

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The human immunoglobulin heavy chain VH region is one of the most complex regions in the human genome. The high level of diversity of this region has been shown by a number of studies. However, because of the limitations of the conventional experimental methods, it has been difficult to learn the extent of the diversity and the underlying mechanisms. This review describes a number of new genetic approaches developed in the authors' laboratory. By using these approaches, significant progress has been made in assigning different VH sequences to their respective loci, in learning the diversity of gene segment number and composition among the VH haplotypes, and in learning VH gene segment organization in individual haplotypes. Information obtained toward this direction could help in understanding the mechanisms underlying VH region diversity and the biological impact of the VH region diversity.

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Multiplex Loss of Heterozygosity Analysis by Using Single or Very Few Cells

Cited by 4 publications

References 19 publications

Direct Detection of Insertion/Deletion Polymorphisms in an Autosomal Region by Analyzing High-Density Markers in Individual Spermatozoa

Direct Detection of Insertion/Deletion Polymorphisms in an Autosomal Region by Analyzing High-Density Markers in Individual Spermatozoa

Low Copy Number DNA Template Can Render Polymerase Chain Reaction Error Prone in a Sequence-Dependent Manner

Genetic diversity of the human immunoglobulin heavy chain V_Hregion

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Multiplex Loss of Heterozygosity Analysis by Using Single or Very Few Cells

Cited by 4 publications

References 19 publications

Direct Detection of Insertion/Deletion Polymorphisms in an Autosomal Region by Analyzing High-Density Markers in Individual Spermatozoa

Direct Detection of Insertion/Deletion Polymorphisms in an Autosomal Region by Analyzing High-Density Markers in Individual Spermatozoa

Low Copy Number DNA Template Can Render Polymerase Chain Reaction Error Prone in a Sequence-Dependent Manner

Genetic diversity of the human immunoglobulin heavy chain VHregion

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Resources

About

Genetic diversity of the human immunoglobulin heavy chain V_Hregion