ATSI, Bari (Italy), 4-5 September 2014, Oral Communication Workshop on Advances in the Turin Shroud Investigation 1 Uncovering the Sources of DNA in the Turin Shroud Barcaccia G. 1 , Galla G. 1 , Achilli A. 2 , Olivieri A. 3 , Torroni A. 3 and Fanti G. 4 1 Laboratorio di Genetica e Genomica, DAFNAE – Università di Padova, Viale Università 16, 3520 Legnaro, Italy; 2 Dipartimento di Chimica, Biologia e Biotecnologie, Università di Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy; 3 Dipartimento di Biologia e Biotecnologie "L. Spallanzani", Università di Pavia, Via Ferrata 9, 27100 Pavia, Italy; 4 Dipartimento di Ingegneria Industriale, Università di Padova, Via Venezia 1, 35137 Padova, Italy. The Shroud of Turin is a linen cloth almost five square meters in size that enveloped a man pieced in the side afterwards his death following crucifixion after being beaten, scourged and crowned with thorns. It is the most important relic of Christianity because the Catholic tradition identifies such burial cloth with that where the body of Jesus Christ was enveloped before being placed in a Palestine tomb about 2000 years ago. Dust particles have been collected directly from the linen cloth of the Turin Shroud and also minutely vacuumed from the interspace between the Turin Shroud and the Holland Cloth sewn to it as reinforcement. All these specimens are derived from different filters corresponding to various areas of the Turin Shroud and some of the dust particles have been removed from the filters and supplied to scientists for investigative studies. Genetic analyses of DNA isolated from plant fibers and cell debris or human blood crusts deriving from samples of the Turin Shroud have been postulated several times. In the past decades, hundreds of pollen grains have been identified and classified in terms of genus and species, using both optical and scanning electron microscopes, in order to be compared with geographic areas in which the corresponding plants did originate and now live. Independent cytological observations have clearly demonstrated that the identified geographic areas are fully compatible with the supposed historic paths travelled by the Turin Shroud. In more recent years, molecular results have apparently supported the existence of human DNA. Nevertheless, the DNA as a whole of the Turin Shroud remains still uncovered. Here we document novel and robust genetic data used for taxonomic identification of plant species on the basis of chloroplast DNA (cpDNA) barcodes and molecular reconstruction of human mitochondrial DNA (mtDNA) haplotypes by exploiting short DNA sequences recovered from pollen grains, cell debris and other minuscule organic specimens, such as plant‐derived fibers and blood‐like clots found into the dusts sampled in the Turin Shroud by STURP Members. In particular, the dust particles analyzed in this study belong to different filters of the back of the Turin Shroud, also corresponding to the areas face, hands, buttocks and feet. Our main task dealing with the attribution of DNA residuals to living organisms or fossils of known genetic identity was engaged from a molecular point of view by isolating, amplifying, subcloning and sequencing specific regions of the genome. In particular, we focused our investigations on nuclear markers (rDNA intergenic transcribed spacers, ITS) and extra‐nuclear markers, of both chloroplast (cpDNA barcodes RuBisCO or rbcL, trnH‐psbA, and trnL‐intron) and mitochondrial (mtDNA targets CYB, COI and NADH dehydrogenase‐subunit 5 along with the hypervariable regions HVRI and HVRII, the so‐ called D‐loop) origin for the analysis of plant and human microscopic residuals, respectively. Organic remnants restrained by the dusts of the Turin Shroud allowed us to extract nanograms of DNA that was then amplified by PCR assay and sequenced by NGS technology (i.e., Roche GS FLX sequencing) in order to recover genic and intergenic markers suitable to identify plant species as well as human haplotypes.