**3.1 Case #1**

The skeletonized human remains from the burial of an unknown soldier from World War II (1941 was presumed to be the year of death) were delivered to the forensic biological department of the Republic Bureau of Forensic Medical Expertise. Skeletal remains were represented by a fragment of the humerus, clavicle, a fragment of the temporal bone, the second cervical vertebra and three molars (**Figure 7**).

Bone and teeth powder (300–400 mg and 100–150 respectively) was used for DNA extraction.

DNA was extracted with PrepFiler Forensic DNA Extraction Kit® (Applied Biosystems, USA) using the standard bone extraction protocol according to the

#### **Figure 7.**

*Bone fragments and teeth from the victim burial were provided for DNA analysis. 1–3 –teeth, 4 – 2nd cervical vertebrae, 5 – Humorous bone fragment, 6 – Clavicle, 7 - temporal bone fragment.*

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**Figure 8.**

*Autosomal STR profile from object #1 (tooth).*

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

manufacturer's instruction with several modifications. The bone and teeth powder was preincubated for 18 hours at 56°C in PrepFiler BTATM Lysis Buffer (Applied Biosystems, USA) with the addition of DTT and proteinase K before the main

Individual DNA profiles on autosomal STRs were detected both from tooth #1 and tooth #2 (**Figure 8**) and were found to be identical to the genotypes determined

In addition, similar Y-chromosome DNA profiles were obtained from these DNA

It is known that DNA is not well preserved in the vertebrae. However, the odontoid process of 2nd cerebral vertebrae is a newly discovered DNA source with a better state of preservation and higher effectiveness for further DNA analysis as

A hair sample was provided to our laboratory for comparison with a possible suspect's DNA profile. According to morphological examination, hair belongs to the group of regional human hairs from the upper extremities. The hair follicle was

partially turned off but the vaginal membranes were preserved.

DNA concentration, measured by qPCR with Quantifiler Human DNA Quantification Kit® (Applied Biosystems, USA), was sufficient for further analysis in two teeth (objects 1, 2) and odontoid process of the 2nd cervical vertebrae (object #4) (0.03 ng/μl and 0.021 ng/μl respectively). DNA concentration extracted from 3rd tooth (object #3), humorous fragment (object #5), clavicle (object #6) and temporal bone fragment (object #7) turned out to be below the lower limit of 0.01 ng/μl. For this reason the samples were set aside for further DNA analysis. DNA profiling was performed by both autosomal and Y-STR loci with AmpFLSTR® Identifiler® Plus PCR Amplification Kit and AmpFLSTR™ Yfiler™ PCR Amplification Kit (Applied Biosystems, USA) according to the manufacturer's

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

in STR typing from the odontoid process. (**Figure 9**).

already described in our previous work [12].

extraction procedures.

instructions.

**3.2 Case #2**

samples (**Figures 10** and **11**).

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

manufacturer's instruction with several modifications. The bone and teeth powder was preincubated for 18 hours at 56°C in PrepFiler BTATM Lysis Buffer (Applied Biosystems, USA) with the addition of DTT and proteinase K before the main extraction procedures.

DNA concentration, measured by qPCR with Quantifiler Human DNA Quantification Kit® (Applied Biosystems, USA), was sufficient for further analysis in two teeth (objects 1, 2) and odontoid process of the 2nd cervical vertebrae (object #4) (0.03 ng/μl and 0.021 ng/μl respectively). DNA concentration extracted from 3rd tooth (object #3), humorous fragment (object #5), clavicle (object #6) and temporal bone fragment (object #7) turned out to be below the lower limit of 0.01 ng/μl. For this reason the samples were set aside for further DNA analysis.

DNA profiling was performed by both autosomal and Y-STR loci with AmpFLSTR® Identifiler® Plus PCR Amplification Kit and AmpFLSTR™ Yfiler™ PCR Amplification Kit (Applied Biosystems, USA) according to the manufacturer's instructions.

Individual DNA profiles on autosomal STRs were detected both from tooth #1 and tooth #2 (**Figure 8**) and were found to be identical to the genotypes determined in STR typing from the odontoid process. (**Figure 9**).

In addition, similar Y-chromosome DNA profiles were obtained from these DNA samples (**Figures 10** and **11**).

It is known that DNA is not well preserved in the vertebrae. However, the odontoid process of 2nd cerebral vertebrae is a newly discovered DNA source with a better state of preservation and higher effectiveness for further DNA analysis as already described in our previous work [12].

#### **3.2 Case #2**

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

sequence of the person suspected to be the biological son.

**3. Examples of DNA-profiling in certain cases**

discovered (**Figure 5**).

**3.1 Case #1**

DNA extraction.

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

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

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.

The skeletonized human remains from the burial of an unknown soldier from World War II (1941 was presumed to be the year of death) were delivered to the forensic biological department of the Republic Bureau of Forensic Medical Expertise. Skeletal remains were represented by a fragment of the humerus, clavicle, a fragment of the temporal bone, the second cervical vertebra and three molars (**Figure 7**). Bone and teeth powder (300–400 mg and 100–150 respectively) was used for

DNA was extracted with PrepFiler Forensic DNA Extraction Kit® (Applied Biosystems, USA) using the standard bone extraction protocol according to the

*Bone fragments and teeth from the victim burial were provided for DNA analysis. 1–3 –teeth, 4 – 2nd cervical* 

*vertebrae, 5 – Humorous bone fragment, 6 – Clavicle, 7 - temporal bone fragment.*

**62**

**Figure 7.**

A hair sample was provided to our laboratory for comparison with a possible suspect's DNA profile. According to morphological examination, hair belongs to the group of regional human hairs from the upper extremities. The hair follicle was partially turned off but the vaginal membranes were preserved.

**Figure 8.** *Autosomal STR profile from object #1 (tooth).*

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

#### **Figure 9.**

*Autosomal STR profile from object #4 (the odontoid process of 2nd cervical vertebrae).*

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

DNA extraction was performed from bulb using Chelex-100 in order to avoid DNA loss and DNA concentration was measured by qPCR using the Quantifiler Human DNA Quantification Kit® (Applied Biosystems, USA) which was showed to be outside the lower detectable limit (0.009 ng/μl) for autosomal STR amplification. However, we attempted to establish an autosomal genetic profile. A profile was

*Y-STR profile in DNA sample extracted from the odontoid process of 2nd cervical vertebrae (object #4).*

The identified loci were found to be matched with one of the suspects; however, the data obtained were not sufficient for the identification process. Accordingly, we analyzed hypervariable regions I and II of the mtDNA D-loop (HVS-1 and HVS-2)

Individual sequences from hair were obtained (**Figure 13**) which that was it made it possible fr there to be a comparison to compare with the suspect's mtDNA

According to these results even one hair with a partially preserved bulb can be sufficient to obtain an incomplete autosomal STR profile which can be supplemented with a profile of mitochondrial DNA to enable a comparison with the DNA of suspects.

The laboratory received biological materials from a crime scene after a quarrel between two men who had been drinking alcohol. One of them committed the murder of the other. The offender tried to hide the traces of the crime by the dismemberment of the body and attempted to destroy the traces by burning them. However, he did not take into account that the human body cannot be burned

partially obtained and the male gender identity was verified (**Figure 12**).

with MitoPlex system (Gordiz, Russia).

sequence (**Figure 14**).

**3.3 Case #3**

**Figure 11.**

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

#### **Figure 10.**

*Y-STR profile in DNA sample extracted from the tooth (object #1).*

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

#### **Figure 11.**

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

*Autosomal STR profile from object #4 (the odontoid process of 2nd cervical vertebrae).*

*Y-STR profile in DNA sample extracted from the tooth (object #1).*

**64**

**Figure 10.**

**Figure 9.**

*Y-STR profile in DNA sample extracted from the odontoid process of 2nd cervical vertebrae (object #4).*

DNA extraction was performed from bulb using Chelex-100 in order to avoid DNA loss and DNA concentration was measured by qPCR using the Quantifiler Human DNA Quantification Kit® (Applied Biosystems, USA) which was showed to be outside the lower detectable limit (0.009 ng/μl) for autosomal STR amplification. However, we attempted to establish an autosomal genetic profile. A profile was partially obtained and the male gender identity was verified (**Figure 12**).

The identified loci were found to be matched with one of the suspects; however, the data obtained were not sufficient for the identification process. Accordingly, we analyzed hypervariable regions I and II of the mtDNA D-loop (HVS-1 and HVS-2) with MitoPlex system (Gordiz, Russia).

Individual sequences from hair were obtained (**Figure 13**) which that was it made it possible fr there to be a comparison to compare with the suspect's mtDNA sequence (**Figure 14**).

According to these results even one hair with a partially preserved bulb can be sufficient to obtain an incomplete autosomal STR profile which can be supplemented with a profile of mitochondrial DNA to enable a comparison with the DNA of suspects.

#### **3.3 Case #3**

The laboratory received biological materials from a crime scene after a quarrel between two men who had been drinking alcohol. One of them committed the murder of the other. The offender tried to hide the traces of the crime by the dismemberment of the body and attempted to destroy the traces by burning them. However, he did not take into account that the human body cannot be burned

#### **Figure 12.** *Partial autosomal STR profile obtained from the hair bulb.*

**Figure 13.** *Part of HVS1 sequence for DNA sample extracted from hair bulb.*

quickly. He tried to pull the body out of the fire by the lower limbs, but the upper limbs and head remained in the pit and continued to burn. Then the suspect threw the lower limbs with a part of the pelvic girdle into the river where they were found a day after. The victim and the criminal were identified.

To identify the deceased person, it was necessary to clarify several circumstances based on the morphological and molecular genetic analysis:

1.whether objects discovered at the fireplace were tho0se of a human;


**67**

**Figure 15.**

hat, gloves).

**Figure 14.**

*MAFFT program (https://mafft.cbrc.jp/).*

belonged to a man.

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

However, there were difficulties in identification of the victim because of the absence of a whole body and in addition, the alleged deceased did not have any relatives other than a sister. By way of biological material relating to the deceased man, items of his clothing with his traces were removed from his house (panties, T-shirt,

*Multiple alignment of mtDNA HVS1 sequences in DNA samples extracted from hair and suspect's blood using* 

Forensic scientists recovered several charred objects of biological (human) origin from the fireplace such as liver fragments (object 1) and finger phalanges

DNA was isolated from these objects, as well as from the lower limbs recovered from the river and clothes of the missing man using the PrepFiler Forensic DNA

The DNA concentration was measured by qPCR using Quantifiler DUO DNA Quantification Kit® (Applied Biosystems, USA). By using Quantifiler DUO it was possible to prove that the lower limbs, burned liver fragments and finger's phalange

(object 2) for further DNA analysis (**Figure 15**).

Extraction Kit® (Applied Biosystems, USA).

*A. Burnt liver fragments; B. burnt finger phalanges.*

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

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

#### **Figure 14.**

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

quickly. He tried to pull the body out of the fire by the lower limbs, but the upper limbs and head remained in the pit and continued to burn. Then the suspect threw the lower limbs with a part of the pelvic girdle into the river where they were found

1.whether objects discovered at the fireplace were tho0se of a human;

3.to establish the identity of the remains discovered in the river;

To identify the deceased person, it was necessary to clarify several circumstances

4.to prove the identity of the missing person's genetic profile using the profile

a day after. The victim and the criminal were identified.

*Part of HVS1 sequence for DNA sample extracted from hair bulb.*

*Partial autosomal STR profile obtained from the hair bulb.*

based on the morphological and molecular genetic analysis:

2.to identify the anatomical origins of the remains;

obtained from the items of clothing

**66**

**Figure 12.**

**Figure 13.**

*Multiple alignment of mtDNA HVS1 sequences in DNA samples extracted from hair and suspect's blood using MAFFT program (https://mafft.cbrc.jp/).*

However, there were difficulties in identification of the victim because of the absence of a whole body and in addition, the alleged deceased did not have any relatives other than a sister. By way of biological material relating to the deceased man, items of his clothing with his traces were removed from his house (panties, T-shirt, hat, gloves).

Forensic scientists recovered several charred objects of biological (human) origin from the fireplace such as liver fragments (object 1) and finger phalanges (object 2) for further DNA analysis (**Figure 15**).

DNA was isolated from these objects, as well as from the lower limbs recovered from the river and clothes of the missing man using the PrepFiler Forensic DNA Extraction Kit® (Applied Biosystems, USA).

The DNA concentration was measured by qPCR using Quantifiler DUO DNA Quantification Kit® (Applied Biosystems, USA). By using Quantifiler DUO it was possible to prove that the lower limbs, burned liver fragments and finger's phalange belonged to a man.

**Figure 15.** *A. Burnt liver fragments; B. burnt finger phalanges.*

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

#### **Figure 16.**

*Autosomal STR profiling from the burnt liver fragment.*

**69**

**Figure 19.**

*Cambridge reference sequence (CRS)).*

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

Both autosomal and Y-STR loci profiles were determined using the

AmpFLSTR® Identifiler® Plus PCR Amplification Kit and AmpFlSTR® Yfiler ™ PCR Amplification Kit (Applied Biosystems, USA). At the same time, the profile of autosomal DNA and Y-chromosome DNA were able to be successfully established in the objects removed from the fire pit, which completely coincided with the profiles

*mtDNA HVS1 sequences alignment in DNA samples extracted from burnt liver fragment (1–1), long bone (1–5) and victim's sister buccal swab (rel) (by MAFFT program (https://mafft.cbrc.jp/) compared to the* 

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

*Partial HVS1 sequence for DNA sample extracted from the burnt liver.*

**Figure 18.**

**Figure 17.** *Autosomal STR profiling from the burnt phalange.*

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

**Figure 18.** *Partial HVS1 sequence for DNA sample extracted from the burnt liver.*


#### **Figure 19.**

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

**68**

**Figure 17.**

**Figure 16.**

*Autosomal STR profiling from the burnt phalange.*

*Autosomal STR profiling from the burnt liver fragment.*

*mtDNA HVS1 sequences alignment in DNA samples extracted from burnt liver fragment (1–1), long bone (1–5) and victim's sister buccal swab (rel) (by MAFFT program (https://mafft.cbrc.jp/) compared to the Cambridge reference sequence (CRS)).*

Both autosomal and Y-STR loci profiles were determined using the AmpFLSTR® Identifiler® Plus PCR Amplification Kit and AmpFlSTR® Yfiler ™ PCR Amplification Kit (Applied Biosystems, USA). At the same time, the profile of autosomal DNA and Y-chromosome DNA were able to be successfully established in the objects removed from the fire pit, which completely coincided with the profiles

obtained in the material of the lower extremities, burnt liver fragments (**Figure 16**), burnt finger phalange (**Figure 17**) and the victim's clothes.

According to the preliminary investigation, the victim had a sister so in order to complete human identification, mtDNA analysis was carried out. Hypervariable regions, HVS-1 and HVS-2, of mtDNA D-loop sequencing was performed by the MitoPlex system (Gordiz, Russia). Individual sequences were also determined in the DNA samples extracted from lower limbs, burnt liver fragments and clothing items of the missing man, as well as from the buccal swabs of the woman believed to be the sister of the deceased man (**Figures 18** and **19**).

Thus, the testing that was conducted made it possible not only to identify the deceased man but also to prove the circumstances of the crime committed. This assisted the process of gauging the circumstances of the offending and the seriousness of the crime committed.
