Nucleic acid-based tests for prenatal gender determination
Provided herein are compositions, processes and kits for noninvasive, early determination of fetal sex from, and/or amount of fetal nucleic acid in, an extracellular nucleic acid sample from a pregnant female. Such compositions, processes and kits are useful for detection of low genomic copy numbers of male fetal nucleic acid in a high copy number background of female nucleic acid, thereby determining the sex of a fetus and/or amount of fetal nucleic acid in a sample.
1. A method for identifying the presence or absence of Y-chromosome nucleic acid in extracellular nucleic acid from a pregnant female, which comprises:
(a) contacting under amplification conditions extracellular nucleic acid from a pregnant female with three or more primer sets selected from the group consisting of:
SEQ
SEQ
ID
corresponding
ID
first primer
NO:
second primer
NO:
AGATGGCTCTAGAGAATCCC
104
GCATTTTCCACTGGTATCCC
137
TTTCCTCTCTCTCCACCCC
105
AAAGTTGCCTTTCTGCCTGC
138
AGAGAAGGCGGATTCCTTTG
106
GAAGTTGGGAGAGTTACTCG
139
ACGAAGGGCTACTTCTCTAC
107
CTCTGGCTAGCATCACAATG
136
GAACCTCAGGCTCTTTGTCC
108
TCTATTCTTGCCGAGAGACC
140
ATGACCCCAAAAGCACAGAC
109
TCCCTCTGTGGTACAGAAAC
141
TCCCAGACTGAAATCCCAAG
110
TCGAATTTGATTCCCAGAGG
142
CTGGCCCTACGAATTTGTTG
111
GTCAGGAGATCGAGACAATC;
143
wherein (i) the nucleic acid comprises maternal nucleic acid and fetal nucleic acid, (ii) each primer of each primer set hybridizes to Y-chromosome nucleic acid, and (iii) each primer set consists of a first primer and a corresponding second primer; and
(b) detecting the presence or absence of amplification products from three or more of the primer sets, whereby the presence of Y-chromosome nucleic acid is based on detecting the presence of the amplification products and whereby the absence of Y-chromosome nucleic acid is based on detecting the absence of the amplification products.
2. The method of claim 1 , wherein the extracellular nucleic acid is deoxyribonucleic acid (DNA).
3. The method of claim 1 , wherein the extracellular nucleic acid is ribonucleic acid (RNA).
4. The method of claim 1 , wherein the extracellular nucleic acid is contacted with one or more first primer and corresponding second primer selected from the group consisting of:
SEQ
SEQ
ID
corresponding second
ID
first primer
NO:
primer
NO:
GATACTATGCCACCAAAGGG
135
CTGAATGAAAGTTCAAACGTC
167
CTCTGGCTAGCATCACAATG
136
TACCTTATGGCAGGGACTTG.
168
5. The method of claim 1 , wherein detecting the presence or absence of the amplification products comprises contacting under extension conditions amplification products with an extension oligonucleotide selected from the group consisting of:
SEQ ID NO:
CCAGAATGCGAAACTC
68
CCCATCTCTTCTCAATCC
69
AAACATGCTCACGATCAC
70
GCAGGGACTTGTCGCTAGG
72
AAAAAGCGCAGGTATTTTCTG
73
CAATTGGATCACATTACATCAAA
74
TGAAATCCCAAGACAATGGA
169
GGCCCTACGAATTTGTTGTATTTTT
76
and detecting the presence or absence of extended extension oligonucleotides.
6. The method of claim 4 , wherein detecting the presence or absence of the amplification products comprises contacting under extension conditions amplification products with an extension oligonucleotide selected from the group consisting of:
SEQ ID NO:
ACAGTACCTGTATTTCCTTAG
173
TATGGCAGGGACTTGTCGCTAGG
174
and detecting the presence or absence of extended extension oligonucleotides.
7. The method of claim 5 , wherein the extended extension oligonucleotides are detected by mass spectrometry.
8. The method of claim 6 , wherein the extended extension oligonucleotides are detected by mass spectrometry.
9. The method of claim 1 , wherein the extracellular nucleic acid is from blood serum.
10. The method of claim 1 , wherein the extracellular nucleic acid is from blood plasma.
11. The method of claim 1 , which further comprises determining the sex of the fetus based on the presence or absence of Y-chromosome nucleic acid, whereby the presence of Y-chromosome nucleic acid determines the fetus is male.
12. The method of claim 1 , wherein the first primers or second corresponding primers or the first primers and second corresponding primers include universal tag sequences.
13. The method claim 1 , wherein the extracellular nucleic acid is contacted with four or more of the primer sets.
14. The method claim 1 , wherein the extracellular nucleic acid is contacted with five or more of the primer sets.
15. The method claim 1 , wherein the extracellular nucleic acid is contacted with six or more of the primer sets.
16. The method claim 1 , wherein the extracellular nucleic acid is contacted with seven or more of the primer sets.
17. The method claim 1 , wherein the extracellular nucleic acid is contacted with eight of the primer sets.
18. The method of claim 17 , which further comprises contacting the extracellular nucleic acid with a control primer set under amplification conditions and detecting the presence or absence of a control amplification product from the control primer set, wherein the control primers of the control primer set can hybridize to the maternal nucleic acid.
19. The method of claim 18 , wherein the control primers of the control primer set hybridize to a nucleotide sequence that encodes or controls the expression of an albumin protein.
20. The method of claim 19 , wherein the control primer set comprises the following control primers:
SEQ
SEQ
ID
corresponding
ID
first primer
NO:
second primer
NO:
CAGTATCTTCAGCAGTGTCC
175
GCAAATTCAGTTACTTCATT
176
C.
21. The method of claim 18 , wherein detecting the presence or absence of the control amplification product comprises contacting under extension conditions each control amplification product with a control extension oligonucleotide and detecting extended control extension oligonucleotide.
22. The method of claim 21 , wherein the control extension oligonucleotide has the nucleotide sequence GCAGTGTCCATTTGAAGAT (SEQ ID NO: 71).
23. A kit that comprises three or more primer sets selected from the group consisting of:
SEQ
SEQ
ID
corresponding second
ID
first primer
NO:
primer
NO:
AGATGGCTCTAGAGAATCCC
104
GCATTTTCCACTGGTATCCC
137
TTTCCTCTCTCTCCACCCC
105
AAAGTTGCCTTTCTGCCTGC
138
AGAGAAGGCGGATTCCTTTG
106
GAAGTTGGGAGAGTTACTCG
139
ACGAAGGGCTACTTCTCTAC
107
CTCTGGCTAGCATCACAATG
136
GAACCTCAGGCTCTTTGTCC
108
TCTATTCTTGCCGAGAGACC
140
ATGACCCCAAAAGCACAGAC
109
TCCCTCTGTGGTACAGAAAC
141
TCCCAGACTGAAATCCCAAG
110
TCGAATTTGATTCCCAGAGG
142
CTGGCCCTACGAATTTGTTG
111
GTCAGGAGATCGAGACAATC
143
wherein the primers comprise a 5′ tag sequence.
24. The kit of claim 23 , comprising eight of the primer sets.
25. A method for determining the amount of fetal nucleic acid in a sample of extracellular nucleic acid from a pregnant female, which comprises:
(a) contacting under amplification conditions extracellular nucleic acid from a pregnant female with three or more primer sets selected from the group consisting of:
SEQ
SEQ
ID
corresponding
ID
first primer
NO:
second primer
NO:
AGATGGCTCTAGAGAATCCC
104
GCATTTTCCACTGGTATCCC
137
TTTCCTCTCTCTCCACCCC
105
AAAGTTGCCTTTCTGCCTGC
138
AGAGAAGGCGGATTCCTTTG
106
GAAGTTGGGAGAGTTACTCG
139
ACGAAGGGCTACTTCTCTAC
107
CTCTGGCTAGCATCACAATG
136
GAACCTCAGGCTCTTTGTCC
108
TCTATTCTTGCCGAGAGACC
140
ATGACCCCAAAAGCACAGAC
109
TCCCTCTGTGGTACAGAAAC
141
TCCCAGACTGAAATCCCAAG
110
TCGAATTTGATTCCCAGAGG
142
CTGGCCCTACGAATTTGTTG
111
GTCAGGAGATCGAGACAATC;
143
(b) detecting the amount of amplification products from three or more of the primer sets, whereby the amount of fetal nucleic acid in the extracellular nucleic acid is determined based on the amount of the amplification product.
26. The method of claim 25 , wherein detecting the amount of the amplification products comprises contacting under extension conditions amplification products with an extension oligonucleotide selected from the group consisting of:
SEQ ID NO:
CCAGAATGCGAAACTC
68
CCCATCTCTTCTCAATCC
69
AAACATGCTCACGATCAC
70
GCAGGGACTTGTCGCTAGG
72
AAAAAGCGCAGGTATTTTCTG
73
CAATTGGATCACATTACATCAAA
74
TGAAATCCCAAGACAATGGA
169
GGCCCTACGAATTTGTTGTATTTTT
76
and detecting the amount of extended extension oligonucleotides.
27. The kit of claim 23 , wherein the 5′ tag sequence comprises a universal primer sequence.