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Human skeletal remains: development of DNA extraction and typing methods
Full text linkNell’ambito delle indagini genetico-forensi i reperti scheletrici sono spesso il solo materiale biologico disponibile per l'identificazione individuale di soggetti scomparsi e rinvenuti in diverse circostanze quali disastri di massa, guerre, eventi socio-politici e accertamenti della paternità biologica effettuati su soggetti deceduti a seguito di esumazione. Il DNA estratto da reperti ossei è normalmente presente in basso numero di copie (Low Copy Number) e altamente degradato a causa di alterazioni chimico-fisiche derivanti sia dalla datazione del reperto biologico stesso sia dalle condizioni ambientali alle quali il campione viene sottoposto talvolta per lunghi periodi di tempo. L’adeguata procedura di estrazione così come l'amplificazione del DNA, rappresentano step fondamentali per l’acquisizione di profili genetici da campioni scheletrici la cui attendibilità è fortemente influenzata dall’integrità del campione.
Per quanto riguarda i resti scheletrici, allo stato attuale non esiste un metodo di estrazione del DNA infallibile e standardizzato, idoneo per la successiva determinazione del profilo genetico, come pure l’amplificazione del DNA mediante marcatori genetici tradizionali quali gli Short Tandem Repeats (STRs) risulta essere talvolta inefficace nei casi che vedono coinvolto del DNA altamente degradato.
In questo studio sono state analizzate diverse tipologie di resti scheletrici umani la cui datazione variava da pochi mesi a circa 90 anni post mortem, rinvenuti in ambienti differenti e quindi caratterizzati da un variabile stato di conservazione. E’ stato sviluppato un nuovo protocollo di estrazione del DNA, consistente in un primo step di purificazione del campione decalcificato e lisato, con il tradizionale metodo fenolo-cloroformio, atto a separare fisicamente il DNA da proteine e materiale contaminante quale ad esempio terriccio. Successivamente ciascun campione è stato estratto mediante kit di estrazione basati su differenti principi chimico-fisici, per valutare sulla base dei profili genetici ottenuti, quale fosse il più idoneo ed efficace nell’estrazione del DNA e da quale distretto osseo si potesse ottenere un profilo genetico di migliore qualità.
L’associazione tra fenolo cloroformio e uno specifico kit basato su estrazione del DNA mediante colonne cromatografiche, si è rivelato essere il metodo più efficace grazie all’utilizzo del fenolo cloroformio che ha permesso di purificare gli estratti impedendo che detriti di varia natura interferissero con le colonne cromatografiche, occludendole e grazie all’elevata capacità estrattiva del kit in esame.
Inoltre, poiché è noto che l’utilizzo di polimorfismi di dimensioni ridotte (Mini Short Tandem Repeats- MiniSTRs) rispetto agli STRs convenzionali risulta essere estremamente efficace nella determinazione di un profilo genetico da campioni di DNA altamente degradato, i primers di otto marcatori STR ampiamente validati e inclusi in numerosi kit commerciali, sono stati ridisegnati in prossimità della regione altamente ripetuta del marcatore prescelto. Gli otto nuovi MiniSTRs sono stati quindi assemblati e suddivisi in due quadruplexes ottenendo prodotti di PCR di dimensioni inferiori a 130 paia di basi.
Il protocollo di estrazione presentato in questo studio ha fornito risultati positivi nei reperti scheletrici analizzati, con elettivo riferimento ai campioni ossei quali femore, di diversa datazione e stato di conservazione. È inoltre da sottolineare come le condizioni ambientali a cui resti sono stati esposti, hanno avuto una maggiore influenza sulla stato di degradazione del DNA rispetto all'età dei reperti scheletrici stessi. Mediante l’utilizzo di kit commerciali per l’amplificazione del DNA e delle due mini-STR quadruplexes sono stati ottenuti profili genetici costituiti da minimo 12 STR da tutti i campioni di femore analizzati, permettendo il riconoscimento dei soggetto deceduto, mediante la comparazione del profilo genetico ottenuto con quello dei presunti parenti. Il metodo di estrazione del DNA descritto in questo lavoro e l’introduzione di nuovi MiniSTRs in aggiunta ai kit commerciali disponibili, sono risultati essere efficaci per la determinazione di profili genetici da campioni scheletrici caratterizzati da DNA altamente degradato.In forensic cases human remains are often the only biological material available for identification of missing persons or unknown remains found in different circumstances such as mass disasters, wars or socio-political events and to solve paternity issues. DNA extracted from bones is often present in low copy number (LCN) and in various states of degradation due to chemical and physical damages produced by intrinsic and extrinsic bone characteristics. Efficient DNA extraction procedures, as well as accurate DNA amplification, are critical steps involved in the process of successful DNA analysis of skeletal samples. Unfortunately, at present there is not an infallible method to recover DNA from very degraded samples due to variations in DNA yield from larger bone fragments that may be attributed to heterogeneity within a bones.
In this study different types of human bones ranging in age from few months to 90 years post mortem, found in various states of preservation and conserved in different places, were analyzed. We developed a modified silica based spin columns protocol, consisting in an initial separation of DNA from proteins and waste material, by using phenol-chloroform to better purify samples. Moreover, as the recovery of information from these degraded samples is enhanced by the use of smaller PCR products (Mini Short Tandem Repeats) rather than conventional STRs, eight STR markers included in available commercial multiplex PCR kits, were redesigned by moving forward and reverse primers in close proximity to the STR repeat region. Two PCR quadruplexes were assembled to obtain PCR products less than 130 bp in size.
Our modified protocol was successfully employed to extract DNA from long bones of different ages and preservation state. Importantly the use of phenol chloroform consistently increased the amount of DNA that could be extracted from long bones, because it allowed to clean samples preventing that waste material interferes with columns or magnetic beads. Environmental conditions under which remains were exposed, had stronger influence on the state of DNA quality than the age of skeletal remains.
Moreover the use of miniSTRs has proposed here could be used in addition to commercial kits, to increase as much as possible the number of markers analyzed. Using amplification commercial kits and the two new mini-STR quadruplex systems we always obtained genetic profiles of at least 12 STR from DNA typing of femur samples.
The improvement of DNA extraction methods and the inclusion of robust and powerful miniSTR loci in addition to the commercial available kits, are effective solutions for forensic practices of degraded DNA samples because ensure that difficult casework samples with low amounts of degraded DNA can be fully typed
Optimization of DNA extraction and typing from skeletal remains.
No full textOptimization of DNA extraction and typing from skeletal remains
Genetic studies of eight X-STRs in a Northeast Italian population
No full textPopulation genetic parameters of eight X-STR (DXS7132, DXS7423, DXS8378, DXS10074, DXS10101, DXS10134, DXS10135, HPRTB) markers located on the X-chromosome in four closely linkage groups were analysed in 176 unrelated Italian individuals (147 females and 29 males) using Mentype® Argus X-8 PCR Amplification Kit (Biotype). The Chi-square test for genotype distribution showed no significant deviation from Hardy-Weinberg equilibrium (HWE). Several microvariant and rare alleles have been observed in some of the X-STR markers studied. PIC of the eight X-STRs ranged from 0.617 to 0.925
Concordance study and allele frequencies for 5 new European Standard Set (ESS) loci in the North-East Italian population
No full textIn this work we performed a concordance study using four different commercial kits that contain fiv
Forensic evaluation of the Investigator DIPplex typing system
No full textInsertion/deletion polymorphisms (known as InDels) are short biallelic length markers widely spreadthroughout the genome. This kind of markers have aroused the interest of the forensic community inrecent years, even if population data are limited. In order to increase the information about Indels, wereport allele frequencies and statistical parameters of forensic efficiency obtained typing a sample of 200unrelated healthy individuals living in North–East Italy using a panel of 30 insertion/deletionpolymorphisms included in Investigator DIPplex kit (Qiagen, Hideln, Germany)
Evaluation of different STR typing kits on DNA extracted from fingernails of unidentified decomposed cadavers
No full textEvaluation of different STR typing kits on DNA extracted from fingernails of unidentified decomposed cadaver
Human skeletal remains: development of DNA extraction and typing assays
No full textExtraction and successful PCR amplification of DNA from human remains in historical and forensic cases has great importance, but is particularly difficult because the methods employed at present are not always satisfactory. Several of them are in fact complicated and time consuming and none methods has reached on acceptance level such that they are routinely used on a widespread basis. Bone extraction protocols currently employed in forensic laboratories tend to be limited because they often fail to give reproducible results, e.g. in cases where bones are exposed to environmental conditions for a long time. In this study different types of human bones ranging in age from few months to 90 years post mortem, found in various states of preservation and conserved in different places, were analyzed.
Different kind of bone, femur, homerus, tibia, jaw, rib, belonging to different cadavers were analyzed. We established a semi-standardized protocol for DNA extraction from bones, verifying which kind of bone yields the best quality of DNA and evaluating different characteristics such as preservation, place of conservation and age of skeletal remains. A comparison in terms of quality of electrophoretic products, was performed from human skeletal remains considering five different DNA extraction methods and starting from a low amount of bone powder (50 -100 mg). In addition to the traditional phenol–chloroform organic method for DNA extraction, four commercial kits were evaluated: QIAamp® DNA Mini kit (Qiagen, Hilden, Germany), QIAamp® DNA Investigator kit (Qiagen, Hilden, Germany), DNA IQTM System (Promega, Milan, Italy) and PrepFilerTM Forensic DNA Extraction Kit (Applied Biosystems, Foster City, CA). Experiments were also performed with a modified protocols developed by Forensic Genetic Laboratory of University of Verona, consisting in an initial separation of DNA from proteins and waste material, by using phenol-chloroform to better purify samples. The new step was introduced after incubation in lysis buffer of different kit solutions. The phenol-chloroform step allowed to clean samples avoiding that the waste material would interfere with columns or magnetic beads.
Moreover as recovery of information from degraded samples is enhanced by the use of smaller PCR products called miniSTR, primer pairs of eight STR markers, included in available commercially multiplex PCR kits, were redesigned by moving forward and reverse primers in close proximity to the STR repeat region. They were assembled in two PCR-multiplexes to obtain PCR products less than 130 bp in size. The choice of this size was determined by the observation that in degraded samples amelogenin is usually the only marker amplified because it is the lowest in size (106-112 bp). In addition to the new miniSTRs multiplexes all samples were amplify also with two kits widespread in forensic use such as AmpFlSTR® IdentifilerTM PCR Amplification Kit (Applied Biosystems), AmpFlSTR® MiniFilerTM PCR Amplification Kit (Applied Biosystems) and PowerPlex® ESI 17 System (Promega).
The improvement of DNA extraction methods and increasing number of miniSTRs in addition to the commercial available kits, may be effective solutions for forensic practices of degraded DNA samples. The application of the DNA extraction protocol based on the use phenol-chloroform for bones exposed to critical environmental conditions for long periods and for low amounts of bone gave good results. Moreover the use of miniSTRs has proposed here could be used in addition to commercial kits, to increase as much as possible the number of markers analyzed
Season specific influence of projected ocean changes on the response to cadmium of stress-related genes in Mytilus galloprovincialis
Full text linkAnthropogenic inputs of carbon dioxide in the atmosphere are driving ocean warming and acidification. The potential threat represented by these changes for marine species could be amplified in coastal areas, characterized by higher levels of pollutants. In addition, temperate organisms may exhibit a different seasonal tolerance to stressors influenced by fluctuations of environmental and physiological factors. In this study, Mediterranean mussels Mytilus galloprovincialis collected both in summer and winter were exposed to combinations of two temperatures (SST, seasonal surface temperature and SST+5 degrees C) and two levels of pH (8.20 and 7.40) in clean or cadmium contaminated seawater (20 mu g/L Cd). mRNA levels of genes related to metal-induced stress response were investigated, including metallothionein mt-20, heat-shock protein hsp70, superoxide dismutase Cu/Zn-sod, catalase cat, glutathione peroxidase gpx1 and glutathione S-transferase gst-pi. To further elucidate if tissues with different physiological roles could exhibit different responsiveness, such analyses were carried out in digestive gland and in gills of exposed mussels. mt-20 mRNA increase after Cd-exposure was higher in the digestive gland than in the gills, with few modulations by temperature or pH only in the latter. Acidification, alone or in combination with other stressors, increased hsp70 mRNA, with seasonaland tissue-specificities (higher in summer and in digestive gland). Among antioxidants, gpx1 mRNA was affected by Cd in both tissues and seasons, with further modulations due to pH and temperature variation tissueand season-specific; in winter the combination of Cd, warming and acidification affected Cu/Zn-sod both in digestive gland and gills and cat only in gills, while weak seasonal variations were observed for gst-pi transcripts only in digestive gland. The overall results highlighted the importance of considering seasonality and responsiveness of different tissues to predict the effects of sudden changes in environmental parameters on responsiveness to and toxicity of chemicals in marine coastal organisms
Evaluation of deleted region from Yp11.2 of two amelogenin negative related males
No full textThe amelogenin locus is encoded by two single copy genes located on the short arm of X (AMELX, on Xp22.1-22.3) and Y (AMELY, on Yp11.2) chromosomes. Few cases of AMELY deletion have been reported and some studies have described that AMELY dropout is due to a large deletion encompassing AMELY on Yp11.2. In this study, we describe a large deleted region on the short arm of the Y chromosome discovered during routine paternity testing in two related males. The complete absence of AMELY and DYS458 marker and the presence of SRY gene in both samples, induced to conclude that a deletion was occurred in a portion of the short arm of Y chromosome. Twenty Y-specific Short Tagged Sequences (STSs) were selected to delineate the breakpoints of deletion. Seven STS between 4.91 Mb and 7.97 Mb were completely absent. The deletion of 3.06 Mb occurred 1.88 Mb upstream and 1.18 Mb downstream of the amelogenin locus. Considering the consequences of a misidentification of a male sample, the use of Y chromosome markers and SRY gene are always crucial in sex determination in criminal investigation
Population genetic evaluation of 12 X-chromosomal short tandem repeats of Investgator Argus X-12 kit in North-East Italy
No full textShort tandem repeats (STRs) located on X chromosome are suitable in complex kinship cases whenanalysis performed using only autosomal loci does not provide informative results. In this work weevaluated the distribution of 12 X-STRs (DXS10103, DXS8378, DXS7132, DXS10134, DXS10074,DXS10101, DXS10135, DXS7423, DXS10146, DXS10079, HPRTB, DXS10148) using Investigator Argus X-12 PCR Amplification kit in a population sample of 207 unrelated healthy individuals (89 females and 118males) living in North-East Italy. Allele frequencies and parameters of forensic interest were calculatedfor the twelve X-STRs. Some new microvariant and rare alleles in the loci DXS10148, DXS10101,DXS10079 have been detected. The Polymorphism Information Content (PIC) of the 12 X-STRs rangedfrom 0.642 (DXS8378) to 0.942 (DXS10135)
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