Methods and compositions for preparation of nucleic acids

1. A method for isolating gDNA suitable for qPCR from a biological material, the method consisting of:

(a) contacting a sample comprising gDNA with a first solution comprising 0.1-0.3 M sodium hydroxide and 0.25-0.5% SDS (w/v) under conditions and for a time sufficient to degrade a cell wall, a cell membrane, a nuclear membrane, or combinations thereof, and/or to denature the gDNA;

(b) mixing into the solution resulting from step (a) a second solution comprising about 0.2 M Tris pH 7.5 and 3-4.5 M ammonium acetate;

(c) centrifuging the sample at a speed and for a length of time sufficient to clarify the neutralized preparation; and

(d) removing insoluble material from the neutralized and clarified preparation,

whereby a solution of gDNA is produced, which is suitable for qPCR.

2. The method of claim 1 , wherein the sample comprises ground plant tissue, optionally ground leaf tissue, ground seed, ground grain, or any combination thereof.

3. The method of claim 1 , wherein the centrifuging step comprises centrifuging the neutralized and clarified preparation for about 10 minutes at about 3200×g.

4. The method of claim 1 , wherein the removing step comprises transferring a supernatant comprising the gDNA that results from the centrifuging step to a new container.

5. The method of claim 4 , further comprising adjusting the supernatant to 10 mM Tris.

6. The method of claim 1 , wherein the removing step comprising transferring the clarified and neutralized preparation to a new container.

7. The method of claim 6 , further comprising diluting the clarified and neutralized preparation in TE.

8. A method for performing a quantitative polymerase chain reaction (qPCR) of a gDNA sample, the method comprising:

(a) providing a gDNA sample prepared by the method of claim 1 ; and

(b) performing PCR under conditions wherein the C T value can be collected for quantitative analysis, whereby qPCR of the gDNA sample is performed.

9. A method for genotyping a gDNA sample, the method comprising:

(a) providing a gDNA sample prepared by the method of claim 1 ; and

(b) performing a molecular assay using at least two primers and two allele-specific probes, wherein end-point signal levels show zygosity status on a 2-D allelic discrimination plot.

10. The method of claim 9 , wherein the molecular assay comprises a single nucleotide polymorphism (SNP) assay.

11. A method for determining a haplotype of a cell with respect to a gene of interest, the method comprising:

(a) providing a gDNA sample isolated from the cell prepared by the method of claim 1 , wherein the gDNA sample comprises gDNA comprising the gene of interest;

(b) performing quantitative PCR (qPCR) on the gDNA sample with one or more nucleic acid reagents that are designed to discriminate between at least two alleles of the gene of interest; and

(c) analyzing products from the qPCR,

wherein a haplotype of the cell with respect to the gene of interest is determined.

12. The method of claim 11 , wherein the one or more nucleic acid reagents comprise at least two sets of oligonucleotide primers, a first set of which is specific for a first allele of the gene of interest and a second set of which is specific for a second allele of the gene of interest.

13. The method of claim 1 , wherein the second solution further comprises 2.5-10.0 mM EDTA.

14. The method of claim 1 , wherein the second solution further comprises 0.5-2.0% PVP40.

15. The method of claim 1 , wherein the first solution comprises 0.2 M sodium hydroxide.