Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence

(a) Flow-chart depicting optimized single-tube (left) and 96-well (right) alkaline differential extraction methods as indicated by parameter sets 22 and 28 in , respectively. Note: A maximum of 2–3 samples should be extracted at one time to prevent exceeding the intended lysis times while transferring substrates to Spin-X inserts. (b) Flow-chart depicting sperm fraction concentration and clean-up steps for single-tube (left) and 96-well (right) alkaline differential extraction methods as indicated by parameter sets 22 and 28 in , respectively. (c) Flow-chart depicting non-sperm fraction concentration and clean-up steps Note: The volume of glacial acetic acid used to adjust the pH of the non-sperm lysates from the single-tube alkaline extraction should be reduced to 2.7μL to compensate for the lower non-sperm lysate volume in the single-tube method (∼386μL) versus ∼709μL for the 96-well method.

(a) Flow-chart depicting optimized single-tube (left) and 96-well (right) alkaline differential extraction methods as indicated by parameter sets 22 and 28 in , respectively. Note: A maximum of 2–3 samples should be extracted at one time to prevent exceeding the intended lysis times while transferring substrates to Spin-X inserts. (b) Flow-chart depicting sperm fraction concentration and clean-up steps for single-tube (left) and 96-well (right) alkaline differential extraction methods as indicated by parameter sets 22 and 28 in , respectively. (c) Flow-chart depicting non-sperm fraction concentration and clean-up steps Note: The volume of glacial acetic acid used to adjust the pH of the non-sperm lysates from the single-tube alkaline extraction should be reduced to 2.7μL to compensate for the lower non-sperm lysate volume in the single-tube method (∼386μL) versus ∼709μL for the 96-well method.

(a) Flow-chart depicting optimized single-tube (left) and 96-well (right) alkaline differential extraction methods as indicated by parameter sets 22 and 28 in , respectively. Note: A maximum of 2–3 samples should be extracted at one time to prevent exceeding the intended lysis times while transferring substrates to Spin-X inserts. (b) Flow-chart depicting sperm fraction concentration and clean-up steps for single-tube (left) and 96-well (right) alkaline differential extraction methods as indicated by parameter sets 22 and 28 in , respectively. (c) Flow-chart depicting non-sperm fraction concentration and clean-up steps Note: The volume of glacial acetic acid used to adjust the pH of the non-sperm lysates from the single-tube alkaline extraction should be reduced to 2.7μL to compensate for the lower non-sperm lysate volume in the single-tube method (∼386μL) versus ∼709μL for the 96-well method.

(a) AmpFlSTR^® Identifiler™ STR profile from sperm fraction of buccal swab spiked with 1/10μL semen that was extracted with parameter set 22 of the single-tube alkaline differential extraction method. Note: The minor alleles designated with arrows are consistent with the female “victim” type. (b) AmpFlSTR^® Identifiler™ STR profile from standard organic/DTT extracted sperm fraction of buccal swab spiked with 1/10μL semen. Note: The minor alleles designated with arrows are consistent with the female “victim” type. (c) AmpFlSTR^® Identifiler™ STR profile from sperm fraction of buccal swab spiked with 1/10μL semen that was extracted with parameter set 26 of the 96-well alkaline differential extraction method. Note: The minor 15 allele designated with an arrow is consistent with the female “victim” 13,15 D8S1179 type. (d) AmpFlSTR^® Identifiler™ STR profile from sperm fraction of buccal swab spiked with 1/10μL semen that was extracted with parameter set 27 of the 96-well alkaline differential extraction method.

(a) AmpFlSTR^® Identifiler™ STR profile from sperm fraction of buccal swab spiked with 1/10μL semen that was extracted with parameter set 22 of the single-tube alkaline differential extraction method. Note: The minor alleles designated with arrows are consistent with the female “victim” type. (b) AmpFlSTR^® Identifiler™ STR profile from standard organic/DTT extracted sperm fraction of buccal swab spiked with 1/10μL semen. Note: The minor alleles designated with arrows are consistent with the female “victim” type. (c) AmpFlSTR^® Identifiler™ STR profile from sperm fraction of buccal swab spiked with 1/10μL semen that was extracted with parameter set 26 of the 96-well alkaline differential extraction method. Note: The minor 15 allele designated with an arrow is consistent with the female “victim” 13,15 D8S1179 type. (d) AmpFlSTR^® Identifiler™ STR profile from sperm fraction of buccal swab spiked with 1/10μL semen that was extracted with parameter set 27 of the 96-well alkaline differential extraction method.

(a) AmpFlSTR^® Identifiler™ STRs from sperm fraction of mock SAE swab containing 1μL semen that was re-extracted with one TE⁻⁴ buffer rinse in place of the 0.1N NaOH non-sperm lysis step in the single-tube alkaline differential extraction (parameter set 18) after it had been processed with the SEB/ProK steps utilized in the DTT differential extraction. Note: The minor alleles designated with arrows are consistent with the female “victim” type and the 15 allele at D8S1179 appears to be elevated stutter at 10.5% of the 16 allele peak height. (b) AmpFlSTR^® Identifiler™ STRs from sperm fraction of mock SAE swab containing 1μL semen that was re-extracted with two TE⁻⁴ buffer rinses in place of the 0.1N NaOH non-sperm lysis step in the single-tube alkaline differential extraction (parameter set 18) after it had been processed with the SEB/ProK steps utilized in the DTT differential extraction. Note: The minor alleles designated with arrows are consistent with the female “victim” type.

(a) AmpFlSTR^® Identifiler™ STRs from sperm fraction of vaginal swab taken 24h post-coitus that was extracted with parameter set 28 of the 96-well NaOH–DNase differential extraction method and typed within 24h from time of sampling. Note: The minor 16 allele designated with an arrow is consistent with the female “victim” 16,16 D19S433 type. (b) AmpFlSTR^® Identifiler™ STRs from non-sperm fraction of vaginal swab taken 24h post-coitus that was extracted with parameter set 28 of the 96-well NaOH–DNase differential extraction method and typed within 24h from time of sampling.