Establishment of Comprehensive Genetic Analysis From a Single Cell

NCT ID: NCT00173732

Last Updated: 2005-09-15

Basic Information

• Recruitment Status: UNKNOWN
• Total Enrollment: 50 participants
• Study Classification: OBSERVATIONAL
• Study Start Date: 2005-09-30

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.

Conditions

Preimplantation Genetic Diagnosis (PGD)

Keywords

Preimplantation genetic diagnosis (PGD); single cell; Multiple displacement amplification (MDA)

Study Design

• Observational Model Type: CASE_CONTROL
• Study Time Perspective: PROSPECTIVE

Eligibility Criteria

Inclusion Criteria

• Preimplantation genetic diagnosis

Exclusion Criteria

• nil

• Minimum Eligible Age: 0 Years
• Eligible Sex: ALL
• Accepts Healthy Volunteers: Yes

Sponsors

National Taiwan University Hospital

OTHER

Sponsor Role: lead

Principal Investigators

Chien-Nan Lee, MD,PhD

Role: STUDY_CHAIR

Locations

Dept of Medical Genetics;National Taiwan University Hospital

Taipei, , Taiwan

Countries

Taiwan

Central Contacts

Yi-Ning su, MD,PhD

Role: CONTACT

Phone: 886-2-23123456

Email: ynsu@ntumc.org

Facility Contacts

Yi-Ning su, MD,PhD

Role: primary

Other Identifiers

9461700629

Identifier Type: -

Identifier Source: org_study_id