Research Description

Forensic Molecular Biology is the molecular analysis of biological evidence to provide objective information on legal matters or those that pertain to criminal and civil law.  The different types of biological evidence may include human bodily fluids or tissues and/or non-human materials such as botanical, fungal, entomological, microbiological or zoological specimens.   Comparison of DNA profiles generated from the biological evidence at the crime scene to known samples can serve to link suspects with crime scenes or victims or can exonerate the suspect.    Forensic molecular biologists use a variety of tools and techniques to examine and characterize biological evidence.  These include microscopy, presumptive chemical testing, immunological analyses, genotyping of autosomal and Y chromosome micro satellite multiplexes (or short tandem repeats -STR), sequencing of mitochondrial DNA hypervariable regions I and II and more recently the use of single nucleotide polymorphism for ethnic profiling and messenger RNA for tissue typing.  Results of the STR tests can be compared to unknowns or to a national DNA database, CODIS (Combined DNA Index System) that currently contains over 2.5 million profiles.  These comparisons can lead to cold hits that provide the ability to solve crimes in cases in which there are no known suspects.

Proposed Undergraduate Role in the Investigation

Dr. Lee (right) with his research students.

Research in my laboratory has fallen into three general categories:

 1. DNA sample collection, archiving and storage strategies for genetic repositories and databases.   Forensic DNA, epidemiological, clinical and any molecular laboratory must store samples of DNA. In these laboratories there is always the possibility that any stored DNA sample may need to be re-tested.  This is especially important when the amount of sample is limited.   Forensic biologists must devise strategies of sampling, testing, preservation and storage to maximize information while minimizing consumption.  In addition to sample quantity, intrinsic differences in sample types resulting in differences in quality, extrinsic differences in the storage buffers especially ionic strength, tube composition and surface type, exposure to UV and temperature and humidity of storage may lead to differences in the ability to recover and re-test the sample.

 The projects in this category aim to evaluate and compare different materials and methods used in DNA storage.  Students in my laboratory have already started testing 4 different tube types at 3 different temperatures over several months of storage.  Results indicate that some polypropylene tubes contain substances that may denature the genetic material that could lead to reduced stability (especially if in the presence of low amounts of single stranded nucleases).  A rapid, fluorescent-based assay for denaturation was developed and tested.  In addition, samples that were stored in a frost-free freezer were less stable that those samples stored at room temperature, presumably due to the freeze-thaw cycles which occur in the frost free freezers.  Preliminary results of this work has been presented and is now in preparation for publication

2.  Y chromosome marker evaluation and testing 

The Attorney General's report to Congress on April 2, 2004 (http://www.ojp.usdoj.gov/nij/sciencetech/dna_pub.htm) found approximately 542,700 criminal cases with biological evidence awaiting DNA testing. These include 52,000 homicide and 169,000 rape cases.   Sexual assault samples have traditionally been screened for the presence of male sample using three methods, acid phosphatase (AP) found in high levels in semen, immunoassays using prostate specific antigen p30 (P 30) and microscopy.  These methods are limited in that they may produce either false positives or false negatives or are time-consuming and tedious.  The development of a male DNA specific screening tool would dramatically improve the processing time of these cases.

Interest in the development of sensitive Y chromosome genetic tests is well supported not only due to the fact that the majority of violent crime is committed by men but also the following reasons: 1. Male cells present may be very small in azoospermic or oligospermic rapists or as in the case of oral copulation only trace epithelial cells may be left, 2. The total number of male cells is low due to loss of sample or degradation, 3. The need to determine the number of semen donors in a multiple rape case requires additional markers, 4. In criminal paternity or mass disaster victim identification (such as the Katrina hurricane disaster), determination of the haplotype of a missing individual may be conducted by a comparison to a male relative (e.g. father, brother, uncle etc), 5. In sexual assaults the time-consuming and sometimes inefficient procedure of differential extraction for the separation of sperm and non-sperm fractions may be by passed and 6. Y STR typing may provide increased statistical discrimination in mixture or kinship analysis cases in which that obtained from autosomal markers is insufficient.   The projects in this category include the collection, extraction, amplification and typing of different populations using Y STR multiplexes for use in the inference of probability of discrimination in different male populations, the development of a Y-alu molecular beacon screening protocol for sexual assault, and the evaluation of Y chromosome single nucleotide polymorphisms using different analytical platforms for forensic biometrics- the ability to determine the likely physical characteristics of an individual by typing genetic markers.   Samples from over 100 individuals of Hispanic, African-american, and Caucasian descent have been collected, extracted and quantified.  Other population samples including 30 Cuban Americans have also been collected and stored at -20C.  Amplifications have been initiated using different Y STR multiplexes.  Results of the genotyping and subsequent analyses will include estimation of haplotype frequencies and may be utilized in phylogenetic analysis for subgroups within each population.

3. Forensic DNA PCR optimization

 Different conditions during amplification may lead to varying levels of quality in the results.  Some of the factors that my alter the quality include the temperature of annealing, the concentration and quality of the template, the ionic composition of the amplification master mix, and the presence of PCR inhibitors or contaminating DNA.  Amplification of STRs from biological evidence may result in the generation of repeat slippage products (also known as stutter) due putatively to slipped strand mispairing.  In this mechanism, a region of the primer and template partially extended duplex denatures and then reanneals out-of register with non-base paired loops of single or multiple repeats being formed on either primer or template strands.  The consequence of this may be a product that is shorter or longer than the template strand.   In the forensic genetic markers, most of the products are 1 repeat shorter. This mechanism is also implicated in neuromuscular disorders such as Huntington's disease and Fragile X as triplet-repeat expansion. Lower levels or elimination of repeat slippage may assist in the interpretation of mixtures as repeat slippage of one of the donor's alleles may occur in the same size as the true allele of the other donor. 

Another set of factors that may influence the results of PCR are the presence of inhibitors. Some known inhibitors include heme (hematin) in blood samples, melanin in tissues and hair, polysaccharides and bile in feces, humic compounds in soil, urea in urine and dyes in denim. 

 Research projects in this area in my laboratory have not yet been initiated thus providing an area for undergraduates to develop and test their own hypotheses.