PCR from single cells for preimplantation diagnosis

Area of Science:

• Human Genetics
• Reproductive Medicine
• Molecular Biology

Background:

• Preimplantation genetic diagnosis (PGD) allows for the selection of unaffected embryos in couples at risk of transmitting inherited disorders.
• This method avoids pregnancy termination by enabling early detection of genetic defects in human embryos after in vitro fertilization (IVF).
• Diagnosis typically involves biopsying single cells from embryos and analyzing them using sensitive molecular techniques.

Purpose of the Study:

• To detail the application of nested polymerase chain reaction (PCR) for the detection of single gene defects in human embryos.
• To highlight the effectiveness of PGD in preventing the transmission of inherited disorders.
• To showcase advancements in PGD for conditions like cystic fibrosis and Lesch-Nyhan syndrome.

Main Methods:

• Biopsy of one to two cells (blastomeres) from 8- to 10-cell embryos on day 3 postinsemination.
• Amplification of informative DNA fragments using nested polymerase chain reaction (PCR).
• Nested PCR involves two rounds of amplification to enhance sensitivity and specificity from limited cellular material.

Main Results:

• Nested PCR allows for the amplification of target sequences from as few as one or two DNA copies within a single cell.
• Successful PGD has been demonstrated for X-linked diseases, cystic fibrosis (CFTR gene), and Lesch-Nyhan syndrome (HPRT gene).
• The nested PCR strategy enhances specificity and yield, reducing the impact of nonspecific amplification and contaminants.

Conclusions:

• Nested PCR is a highly sensitive and specific method for preimplantation genetic diagnosis of single gene defects.
• PGD offers a crucial alternative to later-stage prenatal diagnosis and termination for at-risk couples.
• This technique significantly contributes to reproductive options for families with a history of inherited disorders.