Processes and kits for identifying aneuploidy

1. A multiplex method for identifying the presence or absence of an aneuploidy of a target chromosome in a sample from a pregnant female subject, which comprises:

a. providing about 20 to about 56 amplification primer pairs, wherein each amplification primer pair specifically hybridizes with polynucleotides of a nucleotide sequence species set, wherein: i) the polynucleotides of a nucleotide sequence species set are present on two or more different chromosomes at different loci, comprising a target chromosome and one or more reference chromosomes not associated with the aneuploidy; (ii) the polynucleotides of a nucleotide sequence species set differ by one or more mismatch nucleotides; (iii) the polynucleotides of a nucleotide sequence species set are reproducibly amplified by a single pair of amplification primers relative to each other; (iv) each amplified polynucleotide in a nucleotide sequence species set comprises a nucleotide sequence having the one or more mismatch nucleotides and (v) the polynucleotides of the nucleotide sequence species sets have nucleotide sequences corresponding to SEQ ID NOs: 4736, 4808, 4258, 4413, 4537, 4692, 4203, 4077, 4697, 4436, 3869, 4475, 4078, 4396, 4782, 4683, 4279, 3940, 4236, 3865, 4733, 4168, 3818, 4563, 4906, 3807, 3849, 4594, 4022, 4538, 4219, 4548, 4382, 4374, 4444, 4305, 4403, 4307, 4059, 4767, 3994, 4561, 4209, 3850, 4686, 3985, 4608, 3963, 4038, 4948, 4468, 4879, 4196, 3967, 4150 and 4102;

b. contacting in one or more reaction vessels under amplification conditions, extracellular nucleic acid of the sample comprising fetally derived and maternally derived nucleic acid with amplification primer pairs consisting of the about 20 to about 56 amplification primer pairs of (a), wherein about 10 to about 25 of the amplification primer pairs are in each of the one or more reaction vessels, and each primer pair amplifies only a single nucleotide species set in the reaction vessel, thereby producing about 20 to about 56 species sets of amplified polynucleotides;

c. determining the amount of each amplified polynucleotide in each set by detecting the one or more mismatch nucleotides in each amplified polynucleotide by primer extension, sequencing, Q-PCR or mass spectrometry;

d. determining a ratio between the relative amount of (i) an amplified target polynucleotide and (ii) an amplified reference polynucleotide, for each set; and

e. identifying the presence or absence of an aneuploidy of a target chromosome based on the ratios from the about 20 to about 56 species sets of amplified polynucleotides.

2. The method of claim 1 , wherein the extracellular nucleic acid is from blood, blood plasma or blood serum of the pregnant female subject.

3. The method of claim 2 , wherein the extracellular nucleic acid is from a female subject in the first trimester of pregnancy, second trimester of pregnancy or third trimester of pregnancy.

4. The method of claim 1 , wherein the fetal nucleic acid is about 5% to about 40% of the extracellular nucleic acid; or the number of fetal nucleic acid copies is about 10 copies to about 2000 copies of the total extracellular nucleic acid.

5. The method of claim 1 , which comprises enriching the extracellular nucleic acid for fetal nucleic acid.

6. The method of claim 1 , which comprises determining the fetal nucleic acid concentration in the extracellular nucleic acid.

7. The method of claim 1 , wherein the amounts of the amplified polynucleotides in each set vary by up to a value that permits detection of the aneuploidy of a target chromosome with a confidence level of about 95% or more.

8. The method of claim 1 , wherein the amounts of the amplified polynucleotides in each set vary by up to a value that permits detection of the aneuploidy of a target chromosome with a sensitivity of about 90% or more, and a specificity of about 95% or more.

9. The method of claim 1 , wherein the number of sets of amplified polynucleotides is based on (i) the number of sets that provides a 85% or greater sensitivity for determining the absence of the aneuploidy of a target chromosome, (ii) the number of sets that provides a 85% or greater specificity for determining the presence of the aneuploidy of a target chromosome or (i) the number of sets that provides a 85% or greater sensitivity for determining the absence of the aneuploidy of a target chromosome and (ii) the number of sets that provides a 85% or greater specificity for determining the presence of the aneuploidy of a target chromosome.

10. The method of claim 1 , wherein identifying the presence or absence of an aneuploidy of a target chromosome is based on the ratios from about 56 species sets of amplified polynucleotides.

11. The method of claim 1 , wherein detecting the one or more mismatch nucleotides in each amplified polynucleotide in a set is by primer extension.

12. The method of claim 1 , wherein detecting the one or more mismatch nucleotides in each amplified polynucleotide in a set is by sequencing.

13. The method of claim 1 , wherein detecting the one or more mismatch nucleotides in each amplified polynucleotide in a set is by Q-PCR.

14. The method of claim 1 , wherein detecting the one or more mismatch nucleotides in each amplified polynucleotide in a set is by mass spectrometry.

15. The method of claim 1 , wherein 56 amplification primer pairs are provided.

16. The method of claim 1 , wherein the amplification primer pairs are selected from the primer pairs of the group consisting of:

SEQ

ID

NO:

GTACTCAAATCAAATTGGC

5010

GAAAAAAGTGCATGTCTTTG

5011

CCCAGATGAAGGGGTTTTAG

5012

AAGACCAAGATTCAGAAGC

5013

GTGAATTCTTCCCACTTCTC

5014 

GAGAATTAAAATGAACTGAG

5015

GGTACCACTCATCCATAAAC

5016

ACCCTCAGTACCACTATCTC

5017

CAAGGATTCCAGTACTGGAG

5018

CATATTTGTCTGTGTACTTG

5019

GGAATGTTCCACCTTTCTAC

5020

CTTCAAGGCAATCTTTCTCC

5021

ACCAGCTACATCTAGATTAC

5022

AGCTCCGCTTTGATTTCAGG

5023

TTCCTGATGATAATCTTCCC

5024

AACGGCCAGGGTGGACACT

5025

GATGCTTCTAAGGACCATGT

5026

CCATAGCAAGATGAATTCAC

5027

CACCAGTATCAGCAATAGCTT

5028

GTGCCTAAGGACAACTTTTTC

5029

GAACCATGGTTTGGGTTTAC

5030

AATAAAGTCCAGAGTATGGC

5031

ATTCCAAGGGCTATCTCCAC

5032

GCTGGATACCTAATTAATGC

5033

AGGTAGTTCTCTAAGTTAC

5034

ACAAATATTGACAGGCAGCA

5035

GAACAGCTATATTTCAAACCC

5036

CTTCCTGTGAACCTGCTTTC

5037

GAGGCCAATGCAAATGTAGG

5038

CTTATCCTTTGGGTCTTCTC

5039

TTGTGTGTAGGATTATGAGC

5040

GAATGTAGCTGTTGTTAGGG

5041

TCCCTCTCTCCCTGAAAAAG

5042

GACTAGGTTACTGAGCAAGG

5043

TTAGATTGTTATCCCCACT

5044

AAGAACTCCAGGGCTACTTG

5045

TGGTTCTCAACACTGACCAC

5046

ATGTAGGCATTGTAATGAGG

5047

GATAACATAAGATTAGGAAC

5048

GATTATGATGCACTGGCCTG

5049

CTGAAGAAGTGTAAAAATGGC

5050

CTGCTAACTCAGATACCTGC

5051

GTCTCACATCCCATTTACAG

5052

TCAATTAGAAATCTAGTGC

5053

TATATAATACTTAGTTTTGG

5054

TTCCTTTATGGGAGGAGGAG

5055

ATTGGCCAACATCTCAACAG

5056

GTGTGCTTGCCTCCTAATTT

5057

GAAAGTTCTTGTATTAAAAG

5058

TGTTCTTGGTTGACTTTAC

5059

CTTTTACCAGTTATCTTCC

5060

CATGGGCAAACATAATTTGG

5061

CTGTGTCAATGGCACATCTG

5062

ATATTTCAAGTATCACTATG

5063

GCATCAGGACAAACTGATGG

5064

GTGCTATAAAGCTTTAACAAG

5065

GAGGCAACTAGGACTTAAGG

5066

AGATTATGATGCACTGGCCT

5067

GGAAAGTTAGAAGGCCACAC

5068

TTGTTGCTCCAAGTTTAAG

5069

GTTTTCCCATATCTAGATGTC

5070

TACTTAATCCTTTTGCCTC

5071

GGGCTGTTTCAATGAGGGAC

5072

CTTGTATTAAAAGAAGTGG

5073

GGAGTCAAGGGAGCATTTTA

5074

GAGGCAAACATTATACACAC

5075

ACTGAAGTCATTCATTAGG

5076

GCAGGTTCACAGGAAGTTTC

5077

CTGTGAGGCCAATGCAAATG

5078

GTGGCTATGAAAGACAGCCT

5079

GGGAAGATCTTAAAGGGAGC

5080

ACACCACATTTCTACCACTG

5081

TGATACAGAAATGTCAACCC

5082

CTCCCCAAAGTCTCAGATAG

5083

GGAACAGTGTTGATAAAGACT

5084

CCAAATTTTCAAGCAAAGC

5085

GAAGTGGCCTATCAGGTCT

5086

GGACTTTGGCACCCAAGGA

5087

TTCCTACCTCACTTGGCTTC

5088

GTTACACTGCAAAGCATTTC

5089

GGCAAACATAATTTGGATGGG

5090

CTGTGTCAAATATGTGACTG

5091

TTTCAGACCATTTTTGAAC

5092

AAGAGGGAAGATGACTTTTC

5093

CAGAGGGTAGAAGGGAGGC

5094

GAGTTCTAGTTTGGCAAACTT

5095

ATGCTGATGAACCGCACTTC

5096

CTGGGCAACTGTGAAAAGAC

5097

GACCAAAGTGTATACATAG

5098

CCTTTTAAAATATGCACGAG

5099

TAAATGAGCAGAGACTCAAG

5100

AAAGCTTTAACAAGTTGGCG

5101

GTTGTGACTATTGTTATAG

5102

GACTTGAATTTAACTGCTCC

5103

AACTTGCCTTCAAGATCTG

5104

GAAAAAAGTGCATGTCTTTG

5105

GTCTACCAAACTACAATTAG

5106

CTTTCCAAAAACCCACAATC

5107

AGGGCTGCAGGGACAGTAG

5108

TATTTTTATTTCCAATGTAG

5109

TCATCCCCATTTCTCAACTC

5110

GCTGATCAAGGCAGTTTTTC

5111

TTTAGCATTCCCAGACTCAG

5112

ACTGTTATGTACATTATATC

5113

ACCCTCAGTACCACTATCTC

5114

TGTGTGCAAGGCTCTAGAAG

5115

CCAAGGTTGCTTATAAACAG

5116

GTTCTCTAAGTTACCAAAATC

5117

GTATATATAACTCCTGATC

5118

CAGCATAGCTTTAATGGTCC

5119

TCTGTGACACAGAGCATGAG

5120

AACTCCAGGGCTACTTGAAC

5121.