**2.7 Muscles and tissues**

A stable genetic profile was obtained in more than 85% of cases in tissue analysis. However, negative results in most of the unsuccessful cases were associated with the circumstances of the accident (heating or chemical exposures) which led to DNA damage. In such cases, bones with/without soft tissues remnants were provided for analysis to obtain possible DNA profiles.

**59**

*Reliability and Reproducibility of DNA Profiling from Degraded Samples in Forensic Genetics*

Forest, 2 years 100 100 0 90 — — — Forest, 4 years — 80 — — 30 — 30

Water, 2 years 100 80 — 80 — 60 — Water, 12 years 100 — — 80 — — — Field, 1 year 100 100 100 — — — —

**Femur Ribs Teeth Elbow Brachial** 

80 80 0 50 — — 40

— 90 20 — — 50 —

80 — 30 50 — — —

0 0 0 — — — —

**bone**

**Skull bones**

In two cases it was not possible to obtain a genetic profile of almost fresh muscles without any signs of putrefactive changes even after using different DNA extraction methods (classical phenol-chloroform extraction; magnetic particles extraction (PrepFiler Forensic DNA Extraction Kit® (Applied Biosystems, USA) and ionexchange columns (QIAamp DNA Investigator Kit, Qiagen, USA)). These cases were not related to each other and they did not coincide in time. However, common factors that may influence the effectiveness of genetic analysis were hectic lifestyle and abuse of low-quality alcoholic beverages and alcohol substitutes (as it was

*Effectiveness of autosomal STR loci analysis (%) in different bones (based on AmpFLSTR® Identifiler® Plus* 

It is also difficult to obtain genetic profiles from muscles after a fire but our practice shows that good results can be obtained when examining different tissue types. For example, we determined a stable DNA profile in 5 cases of genetic analysis of liver fragments. Therefore, forensic scientists should be very careful when choosing muscles for DNA analysis and, if it is possible, to duplicate results DNA profiles

A stable genetic profile can be obtained in unexpected for DNA analysis samples. And in contrary, bones or muscles that seem to be quite appropriate for DNA extractions at first glance may turn out to be unsuitable for getting DNA profiles as

For example, a fragment of a tubular bone with soft tissues remnants (**Figure 2**) and a second cervical vertebra (**Figure 3**) from the corpse of an unidentified woman were provided for analysis. The duration of death was no more than 5 days and no

At first glance, the objects seemed to be quite suitable for research. After DNA extraction with PrepFiler Forensic DNA Extraction Kit®, DNA concentration was measured by qPCR with Quantifiler Human DNA Quantification Kit® (Applied

*DOI: http://dx.doi.org/10.5772/intechopen.98300*

**2nd cervical vertebrae (odontoid process)**

**Storage conditions, Death duration**

Forest, around 10 years

Exhumation after 10 years burial

World War II victims (over 78 years), 1–2 m under the ground

Heating track (+800

**Table 1.**

C), 2 years

determined later by bone identification).

*PCR Amplification Kit (Applied Biosystems, USA)).*

further described in several practical cases.

from bones and other tissues.

putrefactive changes were noted.

Biosystems, USA).

*Reliability and Reproducibility of DNA Profiling from Degraded Samples in Forensic Genetics DOI: http://dx.doi.org/10.5772/intechopen.98300*


#### **Table 1.**

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

profiles were obtained in 90% of cases of examined bones.

affect the low percentage of obtained stable profiles.

the same or different extraction methods.

**2.2 Lower limb bones**

**2.3 Upper limb bones**

**2.5 Skull fragments**

process and mandible.

**2.6 Other fragments**

has not been established.

**2.7 Muscles and tissues**

**2.4 Teeth**

from rib fragments after fire and in exhumed corpse bones. In 2% of cases a partial profile was obtained, which made it possible to compare the unidentified corpses with close relatives. In 18% of studied cases DNA profile was not determined.

As noted above, femurs were the most frequent type of bones for DNA analysis; rare cases were presented by the tibia. In three cases, bone fragments (cuts) from lower limbs were provided without specifying their localization. Stable genetic

In most cases, bone fragments were presented by the humerus as well as clavicles,

A stable genetic profile was obtained in 61% of cases and 39% of cases were not successful. Moreover, in seven of the positive cases, the profile was obtained cumulatively while studying other bones from the same skeleton. Several types of research show that teeth are the preferred source for DNA extraction [10, 11]. However, the methods proposed differ from those used in our laboratory which may

Skull bone fragments and whole skulls were provided in only 15 cases. Moreover, the whole skull was presented for analysis in only five cases. A stable genetic profile was obtained in 54% of cases, mainly when analyzing the mastoid

Out of 7 cases of sternum analysis, only three cases gave positive results in determination of stable autosomal STR profile. There have been several cases of examination of fragments of the lung, cancellous bone, calcaneus, vertebrae (without specifying localization). The profile was obtained in all indicated cases. When examining the bones of the pelvis and phalanges of the fingers, the genetic profile

Thus, a stable genetic profile was often obtained in long bones of upper and

A stable genetic profile was obtained in more than 85% of cases in tissue analysis. However, negative results in most of the unsuccessful cases were associated with the circumstances of the accident (heating or chemical exposures) which led to DNA damage. In such cases, bones with/without soft tissues remnants were

lower limbs and ribs compared to the other bone fragments (**Table 1**).

provided for analysis to obtain possible DNA profiles.

It i's significant tooworth noting that in 30% of cases of lower limb bones genotyping, STR profiles were obtained only after repeated DNA extraction using

radial bones and ulna. Positive results were obtained in 65% of cases.

**58**

*Effectiveness of autosomal STR loci analysis (%) in different bones (based on AmpFLSTR® Identifiler® Plus PCR Amplification Kit (Applied Biosystems, USA)).*

In two cases it was not possible to obtain a genetic profile of almost fresh muscles without any signs of putrefactive changes even after using different DNA extraction methods (classical phenol-chloroform extraction; magnetic particles extraction (PrepFiler Forensic DNA Extraction Kit® (Applied Biosystems, USA) and ionexchange columns (QIAamp DNA Investigator Kit, Qiagen, USA)). These cases were not related to each other and they did not coincide in time. However, common factors that may influence the effectiveness of genetic analysis were hectic lifestyle and abuse of low-quality alcoholic beverages and alcohol substitutes (as it was determined later by bone identification).

It is also difficult to obtain genetic profiles from muscles after a fire but our practice shows that good results can be obtained when examining different tissue types. For example, we determined a stable DNA profile in 5 cases of genetic analysis of liver fragments. Therefore, forensic scientists should be very careful when choosing muscles for DNA analysis and, if it is possible, to duplicate results DNA profiles from bones and other tissues.

A stable genetic profile can be obtained in unexpected for DNA analysis samples. And in contrary, bones or muscles that seem to be quite appropriate for DNA extractions at first glance may turn out to be unsuitable for getting DNA profiles as further described in several practical cases.

For example, a fragment of a tubular bone with soft tissues remnants (**Figure 2**) and a second cervical vertebra (**Figure 3**) from the corpse of an unidentified woman were provided for analysis. The duration of death was no more than 5 days and no putrefactive changes were noted.

At first glance, the objects seemed to be quite suitable for research. After DNA extraction with PrepFiler Forensic DNA Extraction Kit®, DNA concentration was measured by qPCR with Quantifiler Human DNA Quantification Kit® (Applied Biosystems, USA).

**Figure 2.** *Tubular bone fragment with soft tissue remnants.*

#### **Figure 3.** *2nd cervical vertebrae.*

According to the PCR results, no DNA was detected in muscle fragments, but DNA concentration in samples extracted from both tubular bone and the odontoid process of the 2nd cervical vertebrae was sufficient for further autosomal STR analysis (0.054 ng/μl and 1.5 ng/μl respectively). However, in spite of sufficient DNA concentration, additional amplification peaks for more than 2 alleles were obtained in some short length loci due to probable stutter effects (**Figure 4**) and normal one and two alleles genotypes were detected for other markers.

Stutter effect can be due to the DNA degradation and the predominance of amplification of shorter fragments that was proved by another qPCR method. We determined DNA quantification and degradation degree estimation by the Quantum DNA-Set kit (Eurogen, Russia) by measuring the concentration of different DNA fragment lengths (91 bp, 156 bp and 211 bp) and their ratio. It turned out that the studied DNA was predominantly presented by the short fragments up to 156 bp length, and their content exceeded the content of long fragments (up to 211 bp) at least three times. After that a number of new DNA extraction experiments were carried out and finally a robust genetic profile was obtained.

In contrast to what was said above, a stable result can be obtained in objects that at first glance are unsuitable for research. Thus, the remains of an unidentified

**61**

**Figure 6.**

*Reliability and Reproducibility of DNA Profiling from Degraded Samples in Forensic Genetics*

*Partial electrophoregram of multiple alleles genotypes for autosomal STR-typing the DNA extracted from 2nd* 

body, discovered after a fire, were delivered to the laboratory. Most of the body was burned out completely (bones were burned out to the point of white heat; the skull was destroyed). In addition, body fragments were exposed by water and

*The DNA profile of autosomal STR loci in DNA sample extracted from burnt blood vessel.*

*DOI: http://dx.doi.org/10.5772/intechopen.98300*

**Figure 4.**

**Figure 5.**

*Fragments of blood vessels after a fire.*

*cervical vertebrae.*

*Reliability and Reproducibility of DNA Profiling from Degraded Samples in Forensic Genetics DOI: http://dx.doi.org/10.5772/intechopen.98300*

**Figure 4.**

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

According to the PCR results, no DNA was detected in muscle fragments, but DNA concentration in samples extracted from both tubular bone and the odontoid process of the 2nd cervical vertebrae was sufficient for further autosomal STR analysis (0.054 ng/μl and 1.5 ng/μl respectively). However, in spite of sufficient DNA concentration, additional amplification peaks for more than 2 alleles were obtained in some short length loci due to probable stutter effects (**Figure 4**) and

Stutter effect can be due to the DNA degradation and the predominance of amplification of shorter fragments that was proved by another qPCR method. We determined DNA quantification and degradation degree estimation by the Quantum DNA-Set kit (Eurogen, Russia) by measuring the concentration of different DNA fragment lengths (91 bp, 156 bp and 211 bp) and their ratio. It turned out that the studied DNA was predominantly presented by the short fragments up to 156 bp length, and their content exceeded the content of long fragments (up to 211 bp) at least three times. After that a number of new DNA extraction experiments were carried out and finally a robust genetic profile was obtained.

In contrast to what was said above, a stable result can be obtained in objects that at first glance are unsuitable for research. Thus, the remains of an unidentified

normal one and two alleles genotypes were detected for other markers.

**60**

**Figure 2.**

**Figure 3.**

*2nd cervical vertebrae.*

*Tubular bone fragment with soft tissue remnants.*

*Partial electrophoregram of multiple alleles genotypes for autosomal STR-typing the DNA extracted from 2nd cervical vertebrae.*

#### **Figure 5.**

*Fragments of blood vessels after a fire.*

#### **Figure 6.**

*The DNA profile of autosomal STR loci in DNA sample extracted from burnt blood vessel.*

body, discovered after a fire, were delivered to the laboratory. Most of the body was burned out completely (bones were burned out to the point of white heat; the skull was destroyed). In addition, body fragments were exposed by water and special agents during extinguishment of the fire. However, in the abdominal and chest cavities, a fragment of a large blood vessel with elements of clotted blood was discovered (**Figure 5**).

Despite the obvious degradation, DNA was isolated by the method of magnetic particles and appropriate concentration was determined (0.18 ng/μl). What is more, the female gender was verified and a partial genetic profile was obtained (**Figure 6**). Further identity determination was continued by the comparison with the mtDNA sequence of the person suspected to be the biological son.

Considering the fact that only a partial profile was obtained for autosomal STRs, hypervariable regions of mtDNA were analyzed both in the blood vessel sample and person suspected to be the son, which showed complete a match in both samples so the identification of unknown corpse was determined by two types of DNA markers.
