METHODS FOR TARGETED SEQUENCING OF CELL-FREE DNA

Smart overview of the Invention

The invention introduces methods for enriching multiple target regions within a single reaction volume using cell-free DNA from blood or plasma samples. This is followed by high-throughput sequencing and sequence read analysis. A library of target-specific oligonucleotide primers or probes is utilized for multiplexed target enrichment, facilitating the amplification of numerous nucleic acid regions simultaneously.

Background

Multiplex PCR is a technique that allows the simultaneous amplification of multiple target nucleic acids in a sample. However, the addition of multiple primer pairs can lead to non-target amplification products, such as primer dimers, which limit further analysis. Improved methods are needed to minimize these non-target products, especially for applications like Non-Invasive Prenatal Genetic Diagnosis (NPD), which require high accuracy and low risk.

Applications

The improved multiplex PCR methods have significant implications for prenatal genetic diagnosis. Current methods for detecting chromosomal abnormalities, which can lead to conditions like Down syndrome, often involve invasive procedures with risks. The new methods aim to enhance sensitivity and specificity while reducing time and costs, potentially replacing invasive techniques with non-invasive blood tests.

Methodology

The methods involve contacting a nucleic acid sample with a library of test primers that hybridize to numerous target loci, followed by a primer extension reaction to produce amplified products. This includes determining the presence or absence of target amplicons and sequencing them. The method also involves selecting primers based on their likelihood of forming dimers, optimizing the primer extension process.

Technical Details

The process includes using non-immobilized, non-identical primers that hybridize to human loci, avoiding molecular inversion probes. The reaction conditions involve specific annealing temperatures and times, which are crucial for successful amplification. The method ensures that the annealing temperature is set above the melting temperature of the primers, enhancing the reaction's efficiency and accuracy.