Discuss appropriate collection and storage methods for human remains recovered from mass disaster, with particular consideration to DNA preservation.
Human remains from mass disaster are to contain either soft and hard tissues of only the body and not the clothing, in different conditions depending upon time of duration time of arrival after the disaster even time has elapsed. Technical personnel group qualified and experienced DNA related Forensic Science should accompany the collection team (Alonso et al., 2005, 541). Alternatively a member of the team should be constantly guided by an outstation expert. Details are provided by Budwole et al. (2005) and Allouche et al. (2008).
Depending upon duration of water or moisture being in contact with the soft tissue, it may have putrefied, andor decayed beyond identification. The remnants of any tissue may have got contaminated with a lot of local microbial flora and others. If burning was also associated, the soft tissue may have charred. Therefore any soft tissue is likely to be unfit. But whatever may becomes available, it is collected after excising in many small units and transferred into previously sterilised dry bags of appropriate sizes and resealed immediately with sterile tools only and properly labelled and numbered as the situation needed.
Buccal cavity related tissues, bones, nail, cartilaginous matter, hair are other sources of choice in that order of priority (Alonso et al., 2005) because they are less likely to disintegrate and their DNA less degraded. They are often used by several forensic investigations because DNA in these is generally intact and non-degraded. Butler (2005) presented exhaustive information on these aspects.
When body putrefaction or other environmental insults destroy soft tissue, hard tissues (bone and tooth) are the preferred samples for DNA. However, in the case of bone, a number of inhibitory factors, including calcium, will prevent successful PCR amplification. Give a stepwise overview of one protocol for extraction of DNA from bone that removes these inhibitory factors and gives a sample that is suitable for PCR analysis. Clearly indicate the step(s) that remove inhibitory factors and briefly state the function of the chemicals andor enzymes used in these step(s).
After DNA extraction by standard procedures, the final purification step has to be preceded by addition of aluminium ammonium sulphate to re-suspended DNA sample. This precipitates the calcium and impurities in the sample. The sample is centrifuged and then passed through affinity resin thiopropyl sepharose 6B removes nearly all potential inhibitors including calcium during the next PCR reaction. The resulting DNA is re-precipitated by standard ethanol and treatment followed by resuspension in buffer and irradiated with electron beam radiations -just before PCR reaction. A protocol suggested by Rohland and Hoffreiter (2007, 1756) is as follows removal of dirt with a tissue moistened with HPLC grade water
outer surface cleaned and a small piece cut using a grinding tool without excess heat formation
Sample is powdered finely in a mortarpestle or using a freezer mill.
Up to 500 mg of the powder was taken in 15 ml tube and 10 ml of extraction solution was added. Another blank tube was also prepared. Extraction solution freshly prepared sealed overnight (16-24 hours) in mild agitation in dark if necessary extraction continued at 560 C for 1-3 hours for better yields. Sample was spun at 5000 g to allow debris to settle.
Supernatant was taken into 40 ml binding buffer in a 50 ml conical tube, add 100 ml silica suspension pH adjusted to about 4.0 with 200 ul (100 ul more if necessary later) of 30 and pH adjusted to about 4.0 tubes were closed and sealed with parafilm for incubation with agitation for 3 h in the dark. Tubes were centrifuged at 5000 g for 2 min. Supernatants were taken in a fresh tube placed in a refrigerator until silica binding is complete.
Buffersilica suspension was taken into a fresh 2 ml tube and centrifuged for 15 s seconds at 16,000g supernatant discarded. Silica pellet washed 2 -3 times with 1 ml fresh buffer dried for about 15 minutes with open lids, 50 ml TE buffer added to dried silica for re-suspension by pippetting up and down slowly, centrifuges for 2 minutes at 16000 g. The supernatant was taken out. This re-suspension repeated twice more
The supernatant contains DNA with most impurities taken away by silica..
When a large number of samples are recovered from a small area, particularly if they are severely decomposed or burned, there is a significant risk of cross contamination samples. Furthermore, working with large numbers of samples in the laboratory simultaneously gives further opportunities for cross contamination and the PCR procedure itself is susceptible to becoming cross contaminated by DNA within the laboratory environment. Explain how the occurrence of PCR contamination can be minimised in the laboratory. Would the STR analysis technique detect if DNA samples from two different victims had been analysed simultaneously
The DNA samples are likely to be contaminated with extraneous DNA from the environment in which the DNA analysis by PCR is to be carried out. This can be considerably minimised by strict adherence to the wearing of the pre-sterilized laboratory aprons by the laboratory personnel for every new sample to be processed. For every DNA analytical laboratory a routinely periodical laboratory staff DNA database is prepared. Before arriving any inferences and conclusive report statements on the test DNA samples these data bases must be checked to eliminate the likely errors arising due to co-occurrence of undesirable DNA interferences.
The other likely inclusions during a PCR analysis are often shown to be tube-specific in which all the pre- and final PCR reactions are carried out. For any such work for subsequent crucial significance is to be performed in a Dedicated laboratory as is meant for use in Ancient DNA laboratory.
When reference DNA from the victim is unavailable, kinship analysis is used to reconstruct a victims DNA profile. Explain the principle of this type of which analysis.
Given the profiles below of the husband and son of a victim at six STR loci, what can be determined regarding the possible allelic profiles of the victim at each loci
As the phrase kinship analysis indicates whenever an appropriate source of true genetic origin of the victim is not available, then alternatives have to be chosen. In the present situation reference DNA samples of the victims husband and son are available. On their own value they may not seem likely to lead to near inferential state. But extensive analysis of the human DNA has provided key insight to how these can have promising value in such situations. The principle of this is based on the understanding that there are 3.2 billion G, C, A, andor T nucleotides present in a specific sequence on twenty three pairs of chromosomes in every cell of any human individual equally inherited from each of the two parents (Butler, 2005). The sequence is 99.7 identical. Thus there are certain regions which are very unique to the individual. From the unique regions microsatellites and the single tandem repeats (STRs) offer the distinguishing parameters for any individual. STR typing is used in collecting forensic evidences. Thirteen locations of these regions are examined to determine the frequency of occurrences of such sequences. These regions called the autosomal loci are distributed on 22 pairs of chromosomes because in the twenty third pair is of the sex chromosomes, one of which is the x chromosome from the female parent anf the other is the y chromosome provided by the male parent. The regions as referred in the above when related to the y chromosome are called the Y-STR loci.
The repeats are counted on the thirteen loci to obtain twenty different measurements. Fifty percent of these have come each of the two parents mother and father. In the table provided above, three of the six STR loci in the son match with same three loci of the father of the. The other three are different. Obviously and logically therefore, the STR loci may have come from the mother of the son. The victims loci observations should match with the sons three loci which are different from the fathers loci. The data shown in the table are not conclusive proofs for victims genetic relationship. In instances such X-STR techniques would provide useful evidences (Bobillo et al., 2008 Aquino et al., 2009).
Occasionally, there may be instances where only a mitochondrial DNA sample can be recovered. Give examples of instances where this is likely to be true. Can kinship analysis, as described for the victim in question 4, be performed on such a sample and, if so, what would be the outcome for this victim
Mitochondrial DNA is inherited from maternally inherited and highly conserved through evolution. It is known to be structurally stable, does not degrade easily, and is well protected in these organelles and highly conserved through evolution. Mitochondrial DNA can be used whenever nuclear DNA may not be available such as in bones and hair. Mt DNA extraction yields per cell are more than other normal cells (Bobillo et al. (2008) have used 10 loci from X-STR and obtained conclusive data.
Pyrosequencing is a relatively new technology for sequencing short segments of DNA. Explain the principles of this technique. Can pyrosequencing be used for STR genotyping based on the currently used PCR amplicons
Pyrosequencing is DNA sequencing of a single stranded DNAs (ssDNA, 300 500 base pairs long) simultaneously with synthesis of the complementary strand developed originally by Ronaghi et al. (1996, Nyren, 2007). The principle is the detection of pyrophosphate released as well as the nucleotide at each step by DNA polymerase by the use of a chemiluminiscent enzyme.
When the resolution of DNA analysis by STR genotyping is not possible, pyrosequencing is found useful as shown recently (Divne et al., 2009). But the present limitation of this is that markers have to be developed fresh. Further, Andrasson et al. (2006) appreciated that mitochondrial DNA quantification from DNA mixtures from several individuals. They developed a pyro-sequencing technique in combination with a DNA quantification.
Depending upon duration of water or moisture being in contact with the soft tissue, it may have putrefied, andor decayed beyond identification. The remnants of any tissue may have got contaminated with a lot of local microbial flora and others. If burning was also associated, the soft tissue may have charred. Therefore any soft tissue is likely to be unfit. But whatever may becomes available, it is collected after excising in many small units and transferred into previously sterilised dry bags of appropriate sizes and resealed immediately with sterile tools only and properly labelled and numbered as the situation needed.
Buccal cavity related tissues, bones, nail, cartilaginous matter, hair are other sources of choice in that order of priority (Alonso et al., 2005) because they are less likely to disintegrate and their DNA less degraded. They are often used by several forensic investigations because DNA in these is generally intact and non-degraded. Butler (2005) presented exhaustive information on these aspects.
When body putrefaction or other environmental insults destroy soft tissue, hard tissues (bone and tooth) are the preferred samples for DNA. However, in the case of bone, a number of inhibitory factors, including calcium, will prevent successful PCR amplification. Give a stepwise overview of one protocol for extraction of DNA from bone that removes these inhibitory factors and gives a sample that is suitable for PCR analysis. Clearly indicate the step(s) that remove inhibitory factors and briefly state the function of the chemicals andor enzymes used in these step(s).
After DNA extraction by standard procedures, the final purification step has to be preceded by addition of aluminium ammonium sulphate to re-suspended DNA sample. This precipitates the calcium and impurities in the sample. The sample is centrifuged and then passed through affinity resin thiopropyl sepharose 6B removes nearly all potential inhibitors including calcium during the next PCR reaction. The resulting DNA is re-precipitated by standard ethanol and treatment followed by resuspension in buffer and irradiated with electron beam radiations -just before PCR reaction. A protocol suggested by Rohland and Hoffreiter (2007, 1756) is as follows removal of dirt with a tissue moistened with HPLC grade water
outer surface cleaned and a small piece cut using a grinding tool without excess heat formation
Sample is powdered finely in a mortarpestle or using a freezer mill.
Up to 500 mg of the powder was taken in 15 ml tube and 10 ml of extraction solution was added. Another blank tube was also prepared. Extraction solution freshly prepared sealed overnight (16-24 hours) in mild agitation in dark if necessary extraction continued at 560 C for 1-3 hours for better yields. Sample was spun at 5000 g to allow debris to settle.
Supernatant was taken into 40 ml binding buffer in a 50 ml conical tube, add 100 ml silica suspension pH adjusted to about 4.0 with 200 ul (100 ul more if necessary later) of 30 and pH adjusted to about 4.0 tubes were closed and sealed with parafilm for incubation with agitation for 3 h in the dark. Tubes were centrifuged at 5000 g for 2 min. Supernatants were taken in a fresh tube placed in a refrigerator until silica binding is complete.
Buffersilica suspension was taken into a fresh 2 ml tube and centrifuged for 15 s seconds at 16,000g supernatant discarded. Silica pellet washed 2 -3 times with 1 ml fresh buffer dried for about 15 minutes with open lids, 50 ml TE buffer added to dried silica for re-suspension by pippetting up and down slowly, centrifuges for 2 minutes at 16000 g. The supernatant was taken out. This re-suspension repeated twice more
The supernatant contains DNA with most impurities taken away by silica..
When a large number of samples are recovered from a small area, particularly if they are severely decomposed or burned, there is a significant risk of cross contamination samples. Furthermore, working with large numbers of samples in the laboratory simultaneously gives further opportunities for cross contamination and the PCR procedure itself is susceptible to becoming cross contaminated by DNA within the laboratory environment. Explain how the occurrence of PCR contamination can be minimised in the laboratory. Would the STR analysis technique detect if DNA samples from two different victims had been analysed simultaneously
The DNA samples are likely to be contaminated with extraneous DNA from the environment in which the DNA analysis by PCR is to be carried out. This can be considerably minimised by strict adherence to the wearing of the pre-sterilized laboratory aprons by the laboratory personnel for every new sample to be processed. For every DNA analytical laboratory a routinely periodical laboratory staff DNA database is prepared. Before arriving any inferences and conclusive report statements on the test DNA samples these data bases must be checked to eliminate the likely errors arising due to co-occurrence of undesirable DNA interferences.
The other likely inclusions during a PCR analysis are often shown to be tube-specific in which all the pre- and final PCR reactions are carried out. For any such work for subsequent crucial significance is to be performed in a Dedicated laboratory as is meant for use in Ancient DNA laboratory.
When reference DNA from the victim is unavailable, kinship analysis is used to reconstruct a victims DNA profile. Explain the principle of this type of which analysis.
Given the profiles below of the husband and son of a victim at six STR loci, what can be determined regarding the possible allelic profiles of the victim at each loci
As the phrase kinship analysis indicates whenever an appropriate source of true genetic origin of the victim is not available, then alternatives have to be chosen. In the present situation reference DNA samples of the victims husband and son are available. On their own value they may not seem likely to lead to near inferential state. But extensive analysis of the human DNA has provided key insight to how these can have promising value in such situations. The principle of this is based on the understanding that there are 3.2 billion G, C, A, andor T nucleotides present in a specific sequence on twenty three pairs of chromosomes in every cell of any human individual equally inherited from each of the two parents (Butler, 2005). The sequence is 99.7 identical. Thus there are certain regions which are very unique to the individual. From the unique regions microsatellites and the single tandem repeats (STRs) offer the distinguishing parameters for any individual. STR typing is used in collecting forensic evidences. Thirteen locations of these regions are examined to determine the frequency of occurrences of such sequences. These regions called the autosomal loci are distributed on 22 pairs of chromosomes because in the twenty third pair is of the sex chromosomes, one of which is the x chromosome from the female parent anf the other is the y chromosome provided by the male parent. The regions as referred in the above when related to the y chromosome are called the Y-STR loci.
The repeats are counted on the thirteen loci to obtain twenty different measurements. Fifty percent of these have come each of the two parents mother and father. In the table provided above, three of the six STR loci in the son match with same three loci of the father of the. The other three are different. Obviously and logically therefore, the STR loci may have come from the mother of the son. The victims loci observations should match with the sons three loci which are different from the fathers loci. The data shown in the table are not conclusive proofs for victims genetic relationship. In instances such X-STR techniques would provide useful evidences (Bobillo et al., 2008 Aquino et al., 2009).
Occasionally, there may be instances where only a mitochondrial DNA sample can be recovered. Give examples of instances where this is likely to be true. Can kinship analysis, as described for the victim in question 4, be performed on such a sample and, if so, what would be the outcome for this victim
Mitochondrial DNA is inherited from maternally inherited and highly conserved through evolution. It is known to be structurally stable, does not degrade easily, and is well protected in these organelles and highly conserved through evolution. Mitochondrial DNA can be used whenever nuclear DNA may not be available such as in bones and hair. Mt DNA extraction yields per cell are more than other normal cells (Bobillo et al. (2008) have used 10 loci from X-STR and obtained conclusive data.
Pyrosequencing is a relatively new technology for sequencing short segments of DNA. Explain the principles of this technique. Can pyrosequencing be used for STR genotyping based on the currently used PCR amplicons
Pyrosequencing is DNA sequencing of a single stranded DNAs (ssDNA, 300 500 base pairs long) simultaneously with synthesis of the complementary strand developed originally by Ronaghi et al. (1996, Nyren, 2007). The principle is the detection of pyrophosphate released as well as the nucleotide at each step by DNA polymerase by the use of a chemiluminiscent enzyme.
When the resolution of DNA analysis by STR genotyping is not possible, pyrosequencing is found useful as shown recently (Divne et al., 2009). But the present limitation of this is that markers have to be developed fresh. Further, Andrasson et al. (2006) appreciated that mitochondrial DNA quantification from DNA mixtures from several individuals. They developed a pyro-sequencing technique in combination with a DNA quantification.
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