NONHUMAN DNA TYPING

 

Eventhough human DNA is generally use in identifying and individualizing evidential samples in routine forensic laboratory analysis, nonhuman DNA typing is still to date considered a specialty application. It’s generally associated with a variety of forensic cases such as animal cruelty or even criminal cases where a person can be linked to a crime scene or another person.

The four main type of nonhuman DNA typing includes

  1. Animal DNA typing – e.g. canine and feline
  2. Plant DNA typing
  3. Insect DNA typing (Forensic entomology)
  4. Microbial DNA typing

Animal DNA Typing

Since cats and dogs are the most widely accepted domestic animal their use in forensic cases have been vastly significant. Bodily fluid samples such as blood, tissue and hair have been applied to both criminal and civil cases using both Nuclear and Mitochondrial DNA typing. Depending on the case itself animals too can be termed as victims, suspects and witness. Animal are said to be victims where animal abused has been implemented or life remains of the animals was left somewhere. An animal is said to be a suspect when for example a canine attack a person or other animal in this case fur, swab for saliva or blood from the victim’s clothes or skin can be examined and DNA profiles can be obtained. An animal can be a witness in which the DNA from bodily fluids or hair can aid in connecting a victim’s pet to a crime scene or suspect vice versa. Both canines and felines have been used and deemed admissible in court in a variety of cases as mentioned above. Despite any criminal cases would use human DNA typing animal typing can be a useful aid with respect to civil cases.

Plant DNA typing

DNA typing of trace evidence from plants material can be used in forensic cases to aid in connecting suspects to crime scene or other individual and vice versa. For example, the seeds from a specific tree located near the crime scene can be associative evidence where its material was found on the suspects pick up van. This was evident in the Palo Verde case in 1992 using Random amplified polymorphic DNA. Cannabis sativa STRs have been studied a bit for forensic application w.r.t drug investigations and fiber type in the form of hemp. There are commercial kits available currently with the aid of liquid nitrogen that extract and purify different types of plant material and used for cases.

Insect DNA typing

Insects play a major part in decomposition and colonization of both human and wild life carcasses. Entomologist uses the insect life cycle and behavior to interpret evidence and in homicide cases to determine the time since death also known as post mortem interval. Insects morphologies can be similar among themselves even if they’re from different species this is where DNA typing applies determining between insect species. Insect species found on a carrion’s body have be individualized through DNA analysis. There are commercial available extraction kits specific for insect DNA samples. FTA cards have also been used to extract and store insect DNA samples. The fly species have the most forensic entomological significance and use DNA typing to differentiate between the three main families calliphoridae (blow fly), muscidae (house fly) and sarcophagidae (flesh life) as morphological features can often be a challenge to differentiate. PCR-RFLP technology have been used to differentiate between fly species by amplification and detection of sequence within the internal transcribed spacer (ITS) region of the ribosomal DNA gene repeat. More validation and research studies are still needed for insect DNA typing for worldwide forensic application among the scientific community.

Microbial DNA typing

Microbes can also be DNA typed in different types of soil material for forensic application. In some crime scene soil can be reliable evidence which can link individual to crime scenes and vice versa using different methods. The characterization on the microbes in the soil is crucial for forensic analysis. Sample similarities, bacterial composition differences and changes in bacterial communities were used to see if bacterial DNA typing of soil samples can be effective in forensic investigations. To distinguish between the microbial samples extraction needs to be done first followed by amplification then typing of the soil samples. Chemical components such as humic acid and PCR inhibitors can be a problem when typing but commercial kits are now available to reduce these problems. These techniques can be easily adapted within laboratories with proper training. For the overall success of these methods validation studies are needed.

                                                             PATERNITY TESTING

 

Paternity test has become one of the most popular types of DNA testing. Everyone is born with a unique genetic outline known as DNA. Because the DNA molecular structure and genetic characteristics of a child are inherited from or determined by the DNA structure of the biological mother and father, DNA identification provides a conclusive and definitive way to establish biological relationships. Therefore, DNA testing has become the most accepted method to determine identity within the legal and scientific communities.

What is paternity testing?

Paternity test assesses and determines a full paternal relationship(fatherhood) of a child. It helps discover a biological link between a prospective father and a child.

Why do a paternity test?

Reasons for undergoing a paternity test are as follows

  1. To work out child support payment.
  2. Resolving question paternity (clarity or closure) where the mother of the child is unsure or uncertain who the father of the child is (multiple partners).
  3. To resolve child custody disputes.
  4. To establish an accurate medical history for the child.
  5. To prevent disputes in adoption
  6. To get an official birth certificate with the known father’s name on it.
  7. For immigration purposes.
  8. To receive an inheritance and resolve will arguments.
  9. As a court order for legal reasons.

How do we determine paternity?

To determine if the alleged father is the true biological father, the DNA profiles of the child, mother and alleged father is compared.

The child inherits two different alleles (alternative form of a gene) at each genetic locus one from the mother and one from the biological father. The allele the mom doesn’t have is term in obligate allele, this is the allele the father must have to be a biological match to the child.

Types of paternity test.

  1. Routine paternity: mother, child and alleged father. 2. Motherless paternity: Child and alleged father. 3. Prenatal paternity: Amniocentesis CVS from mother, fetus and alleged father. 4. Absent alleged father: Mother, child, both paternal grandparents. 5. Siblingship studies: Two siblings, mother (if available). 6. Identical twin studies: One set of twins.

Steps in paternity testing

Sample collection

DNA testing for paternity determination usually uses a non-invasive technique which involves taking a sterile buccal swab saliva sample. In some cases, such as prenatal paternity testing venous blood sample is taken from the arm. After this step the sample is package, labelled and stored appropriately. The sample then follows a chain of custody protocol to the DNA testing laboratory where it is check before analysis can take place.

DNA analysis

The sample is analyzed through a series of specialized techniques in which a DNA profile is generated. When the DNA profiles are obtained statistical calculations are determined for comparison purposes through a strict validation and verification process.

Calculating paternity testing?

A paternity or system index (PI) is used to calculate paternity. This is the relative probability that the alleged father and not an unrelated, randomly selected male of the same ethnic background gave the obligate allele to the child.

PI is applied only when the accused man is a match by having the set of obligate alleles.

Another calculation used in paternity is Combined paternity index (CPI) this is the chances of seeing the entire DNA profile from the real father. Paternity is accepted only is CPI > 100. CPI is a measure of strength of the genetic evidence and is an odds ratio, not a probability.

Possible results

  1. The paternity testing proves conclusively that the tested person is not the biological parent of the child. This statement is normally termed as exclusive or exclusion. The probability of paternity is 0%, and it can be 100% certain that one is not the child’s biological father
  2. The probability of paternity is greater than 99.9% and it documents that more than 99.9% of the male population cannot be the biological father of the tested child. This statement is normally termed as not excluded (inclusion).

Conclusion

Paternity testing is very reliable as the results can be easily understood by a non-scientist. It’s also very convenient as home kits are now available on the market that allows the individuals to take the swab sample home and then return it to the corresponding DNA laboratory pending it’s not a legal case. Privacy can also be given if the individual wants to be anonymous in this case a lab number will be given instead of a name. The application of paternity test is a major worldwide DNA testing process and is expected to increase.

Short Tandem Repeats (STR) ANALYSIS

 A DNA segment that appears more than once on the same chromosome is known as a repeat. Human genomes contain 5-10% of such repetitive sequences that occur in tandem or adjacent to each other. These repetitive sequences vary in size and length and show sufficient variability among individuals in a population. Regions of DNA that contain these short repeat segments are known as short tandem repeats. STR is an accordion-like sequence that occurs between genes. These are very important markers for human identity testing within the forensic community.

STR markers are scattered throughout the human genome where they occur 1/10000 nucleotides. The DNA sequence repeated in an STR motif is usually 2 to 7 base pairs with 4 bases being the preferred size in DNA forensic use. These STR based systems offer many advantages compared to earlier DNA typing techniques e.g. Restriction fragment length polymorphism (RFLP). STR systems provide a rapid and sensitive method to evaluate small amounts (1ng) of human DNA. This small amount of DNA needed for STR systems is 50 times less than what is normally required for RFLP analysis. STR analysis often allows DNA analyst to recover a complete DNA profile even with evidential samples that were exposed to unfavorable conditions such as degraded samples. This is in sharp contrast to RFLP systems which requires a large sample size for analysis to generate a complete DNA profile.

STRs and corresponding loci are easily amplified by Polymerase chain reaction (PCR). PCR amplification of many different STR loci performed simultaneously in the same test tube which is termed multiplexing. When STR profile is generated they are routinely interpreted by direct comparison to DNA standards, allelic ladders (an artificial mixture of common alleles present in the human population for a particular STR marker or locus), and reference standards (know DNA profiles from either the victim or suspect). Probability calculations are determined based upon classical population genetic principles.

For STR markers to be effective across various jurisdictions a common set of standardized markers are used. Currently, the forensic scientific community in the USA has established a set of 13 core STR markers which can be entered in a national database known as the Combined DNA Index System (CODIS) which is a collection of DNA profiles from known offenders. The 13 Core short tandem repeat Loci in CODIS are FGA, VWA, CSF1P0, TPOX, THO1, D18351, D163539, D5S818, D13S317, D73820, D8D1179 and D3S1179. New development with STR analysis includes rapid PCR, portable STR analysis, ancestry inference, and mass spectrometric STR typing permitting detection of repeat region sequence variation. Active DNA databases are being created and numerous forensic cases solved today through generating STR profiles with a common set of genetic markers.

 

 

Y STR ANALYSIS

Y- chromosome structure

It’s the second smallest human chromosome with a length of approximately 60 million nucleotides which represent around 2%–3% of a haploid genome. The Y chromosome has no true homologue in the human genome i.e. it does not have any partner chromosome like the other non-sex chromosomes (autosomal). In contrast to the other chromosomes, the Y is poor in genes sometimes termed ‘gene-poor chromosome’, being more than 50% of its sequence composed of repeated elements.

What is Y chromosome STR marker?

With respect to Forensic DNA analysis, Y chromosome marker is only found in males. The Y chromosome markers determine the paternal lineage which is passed on from generation to generation without changing. 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. Typical examples include sexual assault without ejaculation, sexual assault by a vasectomized male, male DNA under the fingernails of a victim, male ‘touch’ DNA on the skin, and the clothing or belongings of a female victim.

Why use Y chromosome STR analysis?

This marker has a crucial role to play in Forensic DNA analysis

  1. Sexual assault cases where the male DNA component (sperm cells is separated from the female DNA component in a mixed evidence sample.
  2. Males that suffer from azoospermia where there is a deficiency of spermatozoa. Analysis can still be done on the multiple regions along the Y chromosome.
  3. Evolutionary and genealogical history both used for tracing human origins through male lineage lines.
  4. Paternity testing where a male child can be linked to his biological father in motherless cases.
  5. Missing person cases using male relatives as reference samples
  6. Migration patterns

Core Y-STR Haplotype

In 1997 a core set of Y-STRs was selected. Population database has been set up with thousands of male haplotypes Core set: – DYS393 DYS19 – DYS391 DYS437 – DYS439 DYS389I/II DYS438 DYS390 – DYS385a/b DYS392

Interpreting Y-STR Profile

Three possible interpretations of the Y-STR profile for Forensics:

  1. Exclusion • Cannot have originated from the same sample
  2. Inconclusive • Insufficient distinguishing power to identify a match or not
  3. Failure to exclude • Sample may be from the same source or any male relative of source

Future of Y Chromosome Testing

Commercial kits make Y-STRs more accessible and well-suited for Forensic DNA laboratories. Additional markers are being tested which will also increase the power of discrimination. New population studies are being done which will increase the DNA database bank. More accurate likelihoods of Y-STR profiles can be calculated through statistical techniques.