METHODS FOR TARGETED SEQUENCING OF CELL-FREE DNA

Smart overview of the Invention

The patent describes methods for enhancing the efficiency of sequencing specific DNA regions from cell-free DNA found in blood or plasma samples. This approach involves using a set of target-specific oligonucleotide primers or probes to enrich multiple regions of interest simultaneously in a single reaction. The enriched DNA regions are then subjected to high-throughput sequencing and sequence read analysis. The invention aims to improve the accuracy and efficiency of DNA analysis, particularly in the context of non-invasive prenatal genetic diagnosis.

Background

Multiplex PCR is a technique used to amplify multiple target nucleic acid sequences simultaneously, which increases assay throughput and optimizes the use of nucleic acid samples. However, the addition of numerous primer pairs in a single PCR reaction can lead to the formation of non-target amplification products, such as primer dimers, which complicate the analysis. This invention seeks to address these challenges by reducing the formation of non-target amplicons, thereby enhancing the utility of multiplex PCR in various applications, including prenatal genetic diagnosis.

Applications in Prenatal Diagnosis

Current prenatal diagnosis methods, such as maternal blood hormone levels, ultrasound, amniocentesis, and chorion villus biopsy, have limitations in terms of accuracy or invasiveness. Non-invasive methods like maternal blood tests often lack precision, while invasive procedures carry risks such as miscarriage. The proposed methods aim to improve non-invasive prenatal diagnosis by analyzing cell-free fetal DNA in maternal blood, offering higher sensitivity and specificity for detecting chromosomal abnormalities and genetic conditions.

Methodology

The invention involves contacting a nucleic acid sample with a library of primers that hybridize to numerous target loci, followed by subjecting the sample to primer extension reactions. This process generates amplified products that include target amplicons, which can be sequenced or hybridized to an array for analysis. The method also includes selecting primers based on their likelihood of forming dimers, ensuring efficient amplification and reducing non-target products.

Technical Considerations

The method can be adapted by varying the number of target loci and primers, adjusting annealing temperatures, and modifying the duration of the annealing step. These parameters are carefully optimized to enhance the specificity and yield of the target amplicons. Additionally, the inclusion of crowding agents like PEG or glycerol in the reaction mixture can further improve the efficiency of the PCR process. The invention provides a flexible framework for high-throughput sequencing applications, particularly in the field of genetic diagnostics.