Ignite Creation Date:
2024-05-05 @ 11:54 AM
Last Modification Date:
2024-10-26 @ 9:16 AM
Study NCT ID:
NCT00173732
Status:
UNKNOWN
Last Update Posted:
2005-09-15
First Post:
2005-09-13
Brief Title:
Establishment of Comprehensive Genetic Analysis From a Single Cell
Sponsor:
National Taiwan University Hospital
Organization:
National Taiwan University Hospital
Study Overview
Official Title:
Establishment of Comprehensive Genetic Analysis From a Single Cell
Status:
UNKNOWN
Status Verified Date:
2005-06
Last Known Status:
NOT_YET_RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Preimplantation genetic diagnosis PGD is the integration of both assisted reproductive technologies and molecular genetic technologies and was shown to improve implantation rate and reduce spontaneous abortions after implantation The principal problems in single cell PCR include amplification failure ADO and contamination Multiple displacement amplification MDA is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA By this approach the diagnosis of gene disorders form single cell will be more accurate and reliable
Detailed Description:
Preimplantation genetic diagnosis PGD for couples at risk of conceptions with serious genetic disorders is firmly established as a valid reproductive option for couples to consider following appropriate genetic counseling The procedure entails a balance of risks between establishing a successful pregnancy and minimizing the risk of misdiagnosis PGD of single gene disorders relies on PCR-based tests performed on single cells polar bodies or blastomeres Despite the use of increasingly robust protocols allele drop-out ADO the failure to amplify one of the two alleles in a heterozygous cell remains a significant problem for diagnosis using single cell PCR In extreme cases ADO can affect 40 of amplifications and has already caused several PGD misdiagnoses
Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity Because DNA yield from human samples is frequently limiting much effort has been invested in developing methods for whole genome amplification WGA by random or degenerate oligonucleotide-primed PCR However existing WGA methods like degenerate oligonucleotideprimed PCR suffer from incomplete coverage and inadequate average DNA size Multiple displacement amplification MDA provides a highly uniform representation across the genome Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4-6 orders of magnitude for PCR-based WGA methods Average product length was 10 kb MDA is an isothermal strand-displacing amplification yielding about 20-30 μg product from as few as 1-10 copies of human genomic DNA Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells MDA-amplified human DNA is useful for several common methods of genetic analysis including genotyping of single nucleotide polymorphisms chromosome painting Southern blotting and restriction fragment length polymorphism analysis subcloning and DNA sequencing MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies forensics diagnostics and long-term sample storage
In this study we will carefully vary reaction conditions in single cell amplifications from isolated peripheral lymphocytes to minimize the rate of ADO Consideration of the causal factors identified during this study should permit the design of PGD protocols that experience little ADO thus improving the accuracy of PGD for single gene disorders
Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity Because DNA yield from human samples is frequently limiting much effort has been invested in developing methods for whole genome amplification WGA by random or degenerate oligonucleotide-primed PCR However existing WGA methods like degenerate oligonucleotideprimed PCR suffer from incomplete coverage and inadequate average DNA size Multiple displacement amplification MDA provides a highly uniform representation across the genome Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4-6 orders of magnitude for PCR-based WGA methods Average product length was 10 kb MDA is an isothermal strand-displacing amplification yielding about 20-30 μg product from as few as 1-10 copies of human genomic DNA Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells MDA-amplified human DNA is useful for several common methods of genetic analysis including genotyping of single nucleotide polymorphisms chromosome painting Southern blotting and restriction fragment length polymorphism analysis subcloning and DNA sequencing MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies forensics diagnostics and long-term sample storage
In this study we will carefully vary reaction conditions in single cell amplifications from isolated peripheral lymphocytes to minimize the rate of ADO Consideration of the causal factors identified during this study should permit the design of PGD protocols that experience little ADO thus improving the accuracy of PGD for single gene disorders
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None