METHODS FOR SIMULTANEOUS AMPLIFICATION OF TARGET LOCI
The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.
1 . A method for preparing a non-naturally composition of amplified DNA from a maternal blood sample or a fraction thereof useful for non-invasive prenatal aneuploidy determination, wherein the maternal blood sample is from a pregnant mother of a fetus, the method comprising:
(a) extracting cell-free DNA from the maternal blood sample or fraction thereof, wherein the cell-free DNA comprises fetal cell-free DNA and maternal cell-free DNA;
(b) preparing a non-naturally composition of amplified DNA by (i) hybridizing a plurality of circularizing probes to the extracted cell-free DNA or DNA derived therefrom, wherein the circularizing probes bind to a plurality of at least 50 different target loci on one or more chromosomes of interest, (ii) obtaining a plurality of circularized probes by ligation, wherein the circularized probes comprise sequences of the target loci, and (iii) amplifying the circularized probes by rolling-circle amplification; and
(c) analyzing the non-naturally composition of amplified DNA to measure the amounts of the target loci on the one or more chromosomes of interest.
2 . The method of claim 1 , wherein one or more of the target loci map to chromosome(s) X, Y, 13, 18, and/or 21, and the method further comprises determining a ploidy state for chromosome(s) X, Y, 13, 18, and/or 21 of the fetus using the measured amounts of the target loci.
3 . The method of claim 1 , wherein one or more of the target loci map to one or more reference chromosomes expected to be disomic.
4 . The method of claim 1 , wherein one or more of the target loci map to chromosome(s) X, Y, 13, 18, and/or 21, and one or more of the target loci map to one or more reference chromosomes expected to be disomic.
5 . The method of claim 1 , wherein the preparing step further comprises digesting the extracted cell-free DNA or DNA derived therefrom with a restriction enzyme.
6 . The method of claim 1 , wherein the preparing step further comprises removing non-circularized probes with an exonuclease.
7 . The method of claim 1 , wherein the analyzing step comprises measuring the amounts of the target loci by genotyping array, by measurement of fluorescently-tagged DNA, or by high-throughput sequencing.
8 . The method of claim 1 , wherein the method further comprises determining a ploidy state of one or more chromosomes of interest of the fetus using the measured amounts of the target loci.
9 . The method of claim 8 , wherein the determining a ploidy state of one or more chromosomes of interest of the fetus comprises counting the amounts of the target loci that map to a given chromosome.
10 . The method of claim 8 , wherein a Z-score is determined using the amounts of the target loci on a chromosome of interest and a reference chromosome, and the Z-score is used to determine the ploidy state of the chromosome of interest.