METHOD FOR HIGH-THROUGHPUT SCREENING OF TRANSGENIC PLANTS
The invention relates to a method for quantifying levels of expression and/or quantifying copy number of a heterologous polynucleotide in a transgenic plant using quantitative or real-time polymerase chain reaction (QPCR or real-time PCR), wherein the real-time PCR is performed using a primer set specific to a heterologous terminator sequence operably linked to the heterologous polynucleotide.
1 . A method of quantifying the level of expression of a heterologous polynucleotide in a transgenic plant or plant cell, the method comprising the steps of:
a. isolating nucleic acids from a transgenic plant or plant cell, wherein the transgenic plant or plant cell comprises a heterologous polynucleotide operably linked to a heterologous terminator sequence; and
b. quantifying the level of expression of the heterologous polynucleotide by real-time reverse transcriptase polymerase chain reaction using a forward primer and a reverse primer, wherein the forward primer and the reverse primer hybridize to the heterologous terminator sequence or the complement thereof.
2 . A method of measuring the copy number of a heterologous polynucleotide in a transgenic plant or plant cell, the method comprising the steps of:
a. isolating nucleic acids from a transgenic plant or plant cell, wherein the transgenic plant or plant cell comprises a heterologous polynucleotide operably linked to a heterologous terminator sequence; and
b. quantifying the copy number of the heterologous polynucleotide by real-time polymerase chain reaction using a forward primer and a reverse primer, wherein the forward primer and the reverse primer hybridize to the heterologous terminator sequence or the complement thereof.
3 . The method of claim 1 , wherein the heterologous terminator sequence is a SB-GKAF terminator sequence.
4 . The method of claim 2 , wherein the heterologous terminator sequence is a SB-GKAF terminator sequence.
5 . (canceled)
6 . (canceled)
7 . (canceled)
8 . The method of claim 1 wherein the quantification of the level of expression of the heterologous polynucleotide of step (b) is done by real-time reverse transcriptase PCR using a probe that hybridizes to the heterologous terminator sequence or the complement thereof.
9 . The method of claim 2 wherein the quantification of the copy number of the heterologous polynucleotide of step (b) is done by real-time PCR using a probe that hybridizes to the heterologous terminator sequence or the complement thereof.
10 . The method of claim 8 , wherein the heterologous terminator comprises a SB-GKAF terminator sequence and the probe hybridizes to the region of the SB-GKAF terminator sequence bounded by the forward primer and the reverse primer.
11 . The method of claim 10 , wherein the forward primer comprises SEQ ID NO:2, the reverse primer comprises SEQ ID NO:3 and the probe comprises SEQ ID NO:4.
12 . The method of claim 10 , wherein the forward primer comprises SEQ ID NO:5, the reverse primer comprises SEQ ID NO:6 and the probe comprises SEQ ID NO:7.
13 . The method of claim 8 , wherein the transgenic plant or plant cell is a maize plant or plant cell.
14 . A method of quantifying the level of expression of at least two heterologous polynucleotides present in at least two transgenic plants or plant cells, the method comprising the steps of:
a. isolating nucleic acids from at least two transgenic plants or plant cells, wherein a first transgenic plant or plant cell comprises a first heterologous polynucleotide operably linked to a heterologous terminator sequence, and wherein a second transgenic plant or plant cell comprises a second heterologous polynucleotide operably linked to the heterologous terminator sequence;
b. optionally, isolating nucleic acids from additional transgenic plants or plant cells, wherein each of the additional transgenic plants or plant cells comprises an additional heterologous polynucleotide operably linked to the heterologous terminator sequence; and
c. quantifying the level of expression of the first heterologous polynucleotide, the second heterologous polynucleotide and the optional additional heterologous polynucleotides by real-time reverse transcriptase polymerase chain reaction using a forward primer and a reverse primer, wherein the forward primer and the reverse primer hybridize to the heterologous terminator sequence or the complement thereof.
15 . The method of claim 14 wherein the steps of isolation of nucleic acids and quantifying the level of expression of heterologous polynucleotides from additional transgenic plants are done for at least one hundred additional transgenic plants or plant cells.
16 . The method of claim 14 , wherein the heterologous terminator comprises a SB-GKAF terminator sequence and the quantifying step uses a probe which hybridizes to the region of the SB-GKAF terminator sequence bounded by the forward primer and the reverse primer.
17 . The method of claim 16 , wherein the forward primer comprises SEQ ID NO:2, the reverse primer comprises SEQ ID NO:3 and the probe comprises SEQ ID NO:4.
18 . The method of claim 16 , wherein the forward primer comprises SEQ ID NO:5, the reverse primer comprises SEQ ID NO:6 and the probe comprises SEQ ID NO:7.
19 . The method of claim 14 , wherein the transgenic plant or plant cell is a maize plant or plant cell.
20 . The method of claim 15 , wherein the transgenic plant or plant cell is a maize plant or plant cell.
21 . The method of claim 9 , wherein the heterologous terminator comprises a SB-GKAF terminator sequence and the probe hybridizes to the region of the SB-GKAF terminator sequence bounded by the forward primer and the reverse primer.
22 . The method of claim 21 , wherein the forward primer comprises SEQ ID NO:2, the reverse primer comprises SEQ ID NO:3 and the probe comprises SEQ ID NO:4.
23 . The method of claim 21 , wherein the forward primer comprises SEQ ID NO:5, the reverse primer comprises SEQ ID NO:6 and the probe comprises SEQ ID NO:7.
24 . The method of claim 9 , wherein the transgenic plant or plant cell is a maize plant or plant cell.