Methods and compositions for determination of vector backbone in a nucleic acid sample
The invention provides methods and compositions for detecting and/or quantifying vector backbone in a nucleic acid preparation comprising a polynucleotide of interest using amplification assays that amplify a junction located between the polynucleotide of interest and the vector backbone, under conditions whereby amplification can occur, wherein the junction comprises a recognition site for a nuclease, and detecting the absence of an amplification product, whereby the absence of the amplification product indicates low or no vector backbone and/or quantifying the amount of amplification product to determine the amount of vector backbone in the nucleic acid preparation.
1. A method of quantifying the amount of vector backbone in a nucleic acid preparation for use in the transformation of a plant cell, wherein the nucleic acid preparation comprises a polynucleotide of interest (POI), the method comprising:
a) performing an amplification reaction to amplify a junction located between the POI and a vector backbone, under conditions whereby amplification can occur to produce an amplification product, wherein the junction comprises a recognition site for a nuclease;
b) quantifying the amount of amplification product in a quantitative amplification assay, wherein the amount of amplification product indicates the amount of vector backbone in said nucleic acid preparation; and
c) contacting the nucleic acid preparation with a plant cell under conditions favorable for transformation, wherein the cell is subsequently transformed.
2. The method of claim 1 , wherein the junction comprises said recognition site linked at one end to a synthetic polynucleotide (SN), and the junction is linked at one end to the POI via the SN and at the other end to the vector backbone via the recognition site and the amplification product comprises the recognition site and at least a portion of the SN.
3. The method of claim 2 , wherein the amplifying comprises hybridizing a first oligonucleotide primer to the SN of the junction and hybridizing a second oligonucleotide primer to the vector backbone and the amplification product comprises the recognition site, at least a portion of the SN, and a portion of the vector backbone.
4. The method of claim 1 , wherein the junction comprises said recognition site linked at one end to a synthetic polynucleotide SN1 and linked at the other end to a synthetic polynucleotide SN2 and the SN1 of the junction is linked to one of the vector backbone or the POI and the SN2 of the junction is linked to the other of the vector backbone or the POI.
5. The method of claim 4 , wherein the amplifying comprises hybridizing a first oligonucleotide primer to the SN1 of the junction and hybridizing a second oligonucleotide primer to the SN2 of the junction and the amplification product comprises the recognition site, at least a portion of SN1 and at least a portion of SN2.
6. The method of claim 4 , wherein the synthetic polynucleotide SN1 and the synthetic polynucleotide SN2 are the same or different.
7. The method of claim 5 , wherein the synthetic polynucleotide SN1 and the synthetic polynucleotide SN2 are the same or different.
8. The method of claim 1 , wherein amplifying comprises amplifying at least about 50 nucleotides of the junction.
9. The method of claim 8 wherein amplifying comprises amplifying about 50 to about 200 nucleotides of the junction.