Figure 2.—

PCR analysis of nuclear and mitochondrial DNA from cloned Drosophila. (a) Analysis of nuclear DNA: primers 5′-ACTGTTTATTGCCCCCTC-3′ and 5′-GTCGTCGAACAAAAGGTG-3′ amplified a 330-bp fragment of white exon 6 present in both w¹¹¹⁸ and the CaSpeR-4 P-element transformation vector used to create the H2AvDGFP strain. Primers 5′-ACATCAAATTGTCTGCGG-3′ and 5′-CGCTCGTTGCAGAATAGT-3′ amplified a 569-bp fragment of the CaSpeR-4 P-element transformation vector present in H2AvDGFP but absent in w¹¹¹⁸due to a deletion from ∼+2100 to +11,000 relative to the w start codon. PCR allowed for the molecular detection of the CaSpeR-4-based transgene in nuclear transplant recipients. Genomic multiplex PCR at the white locus (1.5% agarose gel) is shown: lane 1, H2AvDGFP genomic control DNA exhibits a 569-bp band from the CaSper-4-derived H2AvDGFP P-element transformation vector and a 330-bp band from white exon 6; lane 2, w¹¹¹⁸ genomic control DNA exhibits the 330-bp band but not the 569-bp Casper-4 band; lane 3, w¹¹¹⁸ nuclei injected into the H2AvDGFP host embryo. The 569-bp CaSper-4-derived fragment is absent while the 330-bp w¹¹¹⁸ fragment is present, indicating only w¹¹¹⁸ nuclei in the cloned embryo; lane 4, H2AvDGFP nuclei injected into the w¹¹¹⁸ host embryo. The 569-bp CaSpeR-4-derived fragment and the 330-bp w¹¹¹⁸ fragment are present, indicating H2AvDGFP nuclei in the cloned embryo; lane 5, DNA negative control; lane 6, 100-bp ladder (MBI Fermentas). DNA was extracted from cloned late-stage embryos and adults using a technique modified from Hatton and O'Hare (http://www.bio.ic.ac.uk/research/ohare/t01816.htm). (b) Analysis of mitochondrial DNA: primers 5′-AATAACAAATTTTTAAGCC-3′ and 5′-GAATAGGGGGAATAAATT-3′ amplified a variable region of the mitochondrial genome ∼759 bp in w¹¹¹⁸ and 773 bp in H2AvDGFP, distinguishing host from donor mitochondria. PCR was performed across a variable region of the mitochondrial genome (4% acrylamide gel): lane 1, the H2AvDGFP control amplifies a 773-bp fragment; lane 2, the w¹¹¹⁸ control amplifies a 759-bp fragment; lane 3, w¹¹¹⁸ nuclei injected into the H2AvDGFP host exhibits the 773-bp fragment from the H2AvDGFP host embryo mitochondria; lane 4, H2AvDGFP nuclei injected into the w¹¹¹⁸ host exhibits the 759-bp fragment from the w¹¹¹⁸ host embryo mitochondria; lane 5, DNA negative control; lane 6, 100-bp ladder (MBI Fermentas).