By Prof. Dr. Christian Rittner, Dr. rer. nat. Peter M. Schneider (auth.), Prof. Dr. Christian Rittner, Dr. rer. nat. Peter M. Schneider (eds.)
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Additional info for Advances in Forensic Haemogenetics: 14th Congress of the International Society for Forensic Haemogenetics (Internationale Gesellschaft for forensische Hämogenetik e.V.), Mainz, September 18–21, 1991
The PCR reaction comprised the following: primers at l~M; dATP, dGTP, dCTP and dTTP each at 200~M. 25U Taq polymerase; lx'PARR' buffer (CamBio), in a total volume of 25~1. DNA template amplified included in this mixture varied from lOng of total DNA extracted from blood, to nonquantifiable amounts isolated from sections of single hair shafts. 5 min, for 35 cycles. aliquots of the PCR product were added directly to a second reaction using a pair of primers internal to those used in the first PCR round: M13(-21)H16401 (5'-TGTAAAACGACGACGGCCAGTTGATTTCACGGAGGATGGTG) and L15997 (5'-CACCATTAGCACCCAAAGCT).
Lanes no. 3: female control (2 pl blood) 40 DYZ3 (Fig. 1). DNA from the male controls gave a high yield of the expected 170 bp repeat fragment (lanes 1, 4 and 5), whereas both the female control (lanes 2 and 3) and the forensic sample gave weaker and larger fragments, which served as convenient internal controls. SRY (Fig. 2A). The F9 fragment was amplified in all three reactions, but only the male control (lane 2) gave the SRY-specific 418 bp fragment. ZFY (Fig. 28). The primers used in these reactions amplify fragments of identica!
The former primer is chimaeric comprising the M13(-21) universal sequencing primer sequence at the 5' end plus the H16401 sequence at the 3' end which is complementary to part the mtDNA D-Loop sequence. The latter primer (L15997) was biotinylated at the 5' end. 5~M final concentration of each primer, lxPARR buffer and 20~M final concentration each dNTP in a final volume of 50~1. 5~1 Advances in Forensic Haemogenetics 4 Edited by Ch. Rittner and P. M. Schneider © Springer-Verlag Berlin Heidelberg 1992 27 SEQUENCING REACTIONS The biotinylated PCR product was added to 50~1 LiCl and 50~1 streptavidincoated Dynal~ beads, followed by incubation at 48°C for 15 mins to immobilise the DNA.
Advances in Forensic Haemogenetics: 14th Congress of the International Society for Forensic Haemogenetics (Internationale Gesellschaft for forensische Hämogenetik e.V.), Mainz, September 18–21, 1991 by Prof. Dr. Christian Rittner, Dr. rer. nat. Peter M. Schneider (auth.), Prof. Dr. Christian Rittner, Dr. rer. nat. Peter M. Schneider (eds.)