Molecular Investigation of Biological Samples for Human Identification

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Abstract

The remarkable advances in DNA technologies over the past two decades had an enormous impact on human identification, medical diagnosis, population genetics, and crime scene investigation. Currently, DNA can be used very specifically to discriminate between individuals, using a wide variety of techniques. Depending on the amount of sample and the level of degradation, several techniques can be applied to narrow the likelihood that a particular individual was present at a crime scene. This paper presents a discussion of the investigative procedures of biological samples for human identification. A brief overview of crime scene, various techniques of molecular biology for identification purpose, PCR and the biological samples involved has been provided. Contrary to popular perceptions, DNA profiling in its present form can’t identify an individual. In RFLP analysis, the long DNA helix is cut into a set of fragments. This can be done with one of a number of specific enzymes called restriction endonucleases, which cut the DNA of all humans at specific points in the DNA base sequence. Quantitative estimation of DNA would be performed and the results stated to determine the concentration of DNA .Sequence analysis would be performed for a particular gene to determine the gender from the samples.

Introduction

A crime scene is any physical scene, anywhere that may provide potential evidence to an investigator and some perspective on the crime that may have transpired. It may include a person’s body, any type of building, vehicles, places in the open air or objects found at those locations. Number of people working on the case depends on the type of case. DNA forensics is the study of forensic evidence leading to various activities or happenings. It can be used to identify individuals by the characteristics of their DNA. DNA forensics has also played an important role in present day medical diagnosis, human identification, crime scene investigation etc. DNA forensics can also be used to exonerate individuals, who have been falsely accused of committing crimes,
and to connect two different crimes scenes that may share some evidence .It can also be used to identify the father of a child achieved through rape or incest. In some cases, it can also be used to identify the rapist without the child. Biological samples such as blood, hair, semen etc play an important role in crime scene investigation.
Once the DNA (required amount) is detected , it can further be analysed by using one of the many DNS testing methods such as RFLP analysis , STR analysis , Y-chromosome typing, or mitochondrial DNA (mDNA ) sequence analysis .
The testing method used depends on
 Type of sample
 Amount of sample
 The condition of the sample
Variations in DNA sequence between individuals are termed “polymorphisms”. Sequences with the highest degree of polymorphism are very useful for DNA analysis in forensics cases and paternity testing.
STR technology has enabled the rapid generation of highly informative DNA data for use in human identification. Human review (in duplicate) of forensic STR data is currently required to assure that DNA profiles are correctly scored. However, this human analysis step has become a labor-intensive bottleneck impeding the rapid construction of high-quality DNA databases.’ The practical application of DNA technology to the identification of biological material has had a significant impact on forensic biology, because it enables much stronger conclusions of identity or non-identity to be made. The vast majority of the DNA in a cell has no known function, and this non-coding DNA varies very greatly from one individual to another. This is the DNA which is examined in most DNA profiling systems.
In RFLP analysis, the long DNA helix is cut into a set of fragments. This can be done with one of a number of specific enzymes called restriction endonucleases, which cut the DNA of all humans at specific points in the DNA base sequence. Thus a set of fragments is obtained, one of which contains the
region of interest. This fragment is identified using a short piece of synthetic DNA called a probe. The probe is designed so that its base sequence is complementary to (that is forms A-T and G-C pairs with) the base sequence of one of the two strands in the fragment of interest. It measures fragments of DNA containing short sequences that vary from person to person, called VNTRs.
The technical complexity of performing RFLP analysis coupled with the widespread use of short-lived radioisotopes for detection have prompted researchers to look for alternative methods.
The advent of the PCR has resulted in the development of a large number of molecular techniques, which offer an effective alternative to the hybridization methods of RFLP analysis.
The technical complexity of RFLP analysis coupled with widespread use of radioisotopes for detection have persuaded researchers to look for and apply alternative methods . Advent of PCR has resulted in the development of a large number of molecular techniques alternative to the RFLP analysis .
PCR based approaches have few advantages i.e they use only small amount of DNA and also avoid the use of radioactivity . They are even susceptible to automation. Lineage markers have special applications in forensic genetics. Y chromosome analysis is very helpful in cases where there is an excess of DNA from a female victim and only a low proportion from a male perpetrator. The steady growth in the size of forensic DNA databases raises issues on the criteria of inclusion and retention and doubts on the efficiency, commensurability, and infringement of privacy of such large personal data collections. In contrast to the past, not only serious but all crimes are subject to DNA analysis generating millions and millions of DNA profiles, many of which are stored and continuously searched in national DNA databases.
Extraction of DNA basically consists of four major steps:
•Preparation of a cell extract
•Purification of DNA from cell extract •Concentration of DNA samples
•Measurement of purity and DNA concentration
A new forensic DNA approach that is being more broadly applied is mitochondrial DNA (mtDNA) sequencing. Mitochondria are structures within cells that contain DNA that is distinct from the DNA in the nucleus. Mitochondrial sequencing is used extensively in FBI labs, as well as state and private crime labs. This technique, which takes into account small differences between people’s mtDNA, is very useful for minute samples or ancient or degraded samples. This is because cells are loaded with mitochondria, so even in highly degraded samples or old hair, there is often enough mtDNA to obtain a sequence. DNA extraction and purification is a very important part of DNA forensics.

Conclusion

The above work concludes that the maximum concentration of the DNA obtained was from the Buccal sample. All the other samples were equally quantified for the extracted DNA. However the DNA sequence remains the same whatever is the source of extraction from the same organism. The study was focused on sex determination using the AMEL gene sequencing. The sequence of AMEL was retrieved from NCBI and a specific primer was designed for the amplification using PCR. The amplicon thus obtained was sequenced and the sequence was analyzed for sex determination. The STR profile showed homozygous peaks representing the Female sample. These techniques can be applied for CARIME INVESTIGATION, Disease identification etc.

Bibliography

1) http://www.nature.com/scitable/topicpage/forensics-dna-fingerprinting-and-codis-736
2) http://www.dnaforensics.com/
3) FOR ENSICSAND MITOCHONDRIAL DNA: Applications, Debates, and Foundations*, Bruce Budowle,1 Marc W. Allard,2 Mark R. Wilson,3 and Ranajit Chakraborty4 4) “DNA-based prediction of human externally visible characteristics in forensics: Motivations, scientific challenges, and ethical considerations”, Manfred Kayser et.al.,
5) http://www.crimescene-forensics.com/Crime_Scene_Forensics/DNA.html
6) https://www.forensic.co.in/cyber_crime_investigation/

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