Differential extraction

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Differential extraction (also known as differential lysis) refers to the process by which the DNA from two different types of cells can be extracted without mixing their contents. The most common application of this method is the extraction of DNA from vaginal epithelial cells and sperm cells from sexual assault cases in order to determine the DNA profiles of the victim and the perpetrator. Its success is based on the fact that sperm cells pack their DNA using protamines (rather than histones) which are held together by disulfide bonds. The protamines sequester DNA from spermatozoa, making it more resilient to DNA extraction than DNA from epithelial cells.

After determining that sperm cells are present (typically through staining and light microscopy) in a vaginal/rectal sample, the subject's epithelial cells are lysed by a standard DNA extraction method, like a phenol/chloroform extraction and their DNA extracted through normal means. The epithelial DNA in solution is removed and saved, while the sperm cell's DNA precipitates with the attached protamines. Differential extraction uses a chemical called dithiothreitol (DTT) to disrupt the sulfur bonds in the protamines in order to release its DNA. Once the DNA is detached from the protamines, it is prone to standard DNA extraction methods. This creates two different DNA fractions from one sample, that of the victim and that of the perpetrator.

However, the described method is difficult to carry out because it is both very labor-intensive and time-consuming, leading to a build-up of untested rape kits. An estimated 500.000 rape kits alone in the US. [1] Greenspoon et al. [2] reported improvements in sample processing efficiency and throughput using robotic automation. However, associated costs for implementation in conjunction with low-throughput quantities of samples may be impractical for forensic laboratories and cannot be justified. [3]

Recently a multistep nucleic acid extraction procedure was introduced to provide DNA lysates of highest quality. [4] A self-sealing membrane allows a stepwise release and separation of DNA from mixed specimens. Implemented in a spin-column system, it is ideally suitable for DNA extraction procedures involving differential extraction of forensic samples such as epithelium, saliva or blood vs. sperms. Simple and reliable extraction protocols for both, stained samples as well as gynecological swabs, respectively, overcome the often claimed difficulties in differential extraction (e.g. losing a sperm pellet through several washing steps).

Furthermore, an early qualified decision whether the process of a differential extraction is worth the time and efforts is possible due to gradual buffer separation. As an immunological pre-test for semen in a sample of sexual assault can be carried out using the identical sample.

Workflow

Workflow of the differential lysis of a sample of sexual assault evidence is shown. All steps of the stepwise DNA-extraction process are described in detail. Differential lysis in a single-tube extraction process for accurate forensic profiling.jpg
Workflow of the differential lysis of a sample of sexual assault evidence is shown. All steps of the stepwise DNA-extraction process are described in detail.

First of all human proteins, e.g. human semenogelin antigen, can be optional isolated and immunologically analyzed to quick-check for the presence of human seminal fluid. If this test shows a positive result, the same casework sample will be processed further. [5] The first part of the differential lysis is carried out under mild conditions to gain the female DNA. Lysate of the epithelia cells is retained within the column even during heat incubation and moderate shaking. Upon centrifugation, the solution passes the column into the collection tube. The DNA lysate is further purified and used to generate an autosomal STR profile of the victim. To further obtain the male DNA which will identify the perpetrator, harsher lysis conditions will be applied afterwards to the very same filter column without the necessity of sample carriage in order to break open the retained spermatozoa. Due to the high yield of DNA, the chance of a successful DNA-profile by downstream analysis is significantly increased. This simple handling allows time savings and higher throughput in this manual process to improve crime solution rates and to speed up analysis of backlogged crime samples.

Related Research Articles

<span class="mw-page-title-main">Spermatozoon</span> Motile sperm cell

A spermatozoon is a motile sperm cell produced by male animals relying on internal fertilization. A spermatozoon is a moving form of the haploid cell that is the male gamete that joins with an ovum to form a zygote.

<span class="mw-page-title-main">Intracytoplasmic sperm injection</span> In vitro fertilization procedure

Intracytoplasmic sperm injection is an in vitro fertilization (IVF) procedure in which a single sperm cell is injected directly into the cytoplasm of an egg. This technique is used in order to prepare the gametes for the obtention of embryos that may be transferred to a maternal uterus. With this method, the acrosome reaction is skipped.

Lysis is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a lysate. In molecular biology, biochemistry, and cell biology laboratories, cell cultures may be subjected to lysis in the process of purifying their components, as in protein purification, DNA extraction, RNA extraction, or in purifying organelles.

A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells. Most lysis buffers contain buffering salts and ionic salts to regulate the pH and osmolarity of the lysate. Sometimes detergents are added to break up membrane structures. For lysis buffers targeted at protein extraction, protease inhibitors are often included, and in difficult cases may be almost required. Lysis buffers can be used on both animal and plant tissue cells.

The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR, sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.

Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other electrophoretic techniques including capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis and micellar electrokinetic chromatography (MEKC) belong also to this class of methods. In CE methods, analytes migrate through electrolyte solutions under the influence of an electric field. Analytes can be separated according to ionic mobility and/or partitioning into an alternate phase via non-covalent interactions. Additionally, analytes may be concentrated or "focused" by means of gradients in conductivity and pH.

A rape kit or rape test kit is a package of items used by medical, police or other personnel for gathering and preserving physical evidence following an instance or allegation of sexual assault. The evidence collected from the victim can aid the criminal rape investigation and the prosecution of a suspected assailant. DNA evidence can have tremendous utility for sexual assault investigations and prosecution by identifying offenders, revealing serial offenders through DNA matches across cases, and exonerating those who have been wrongly accused.

A protein microarray is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound. Probe molecules, typically labeled with a fluorescent dye, are added to the array. Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner. Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents. The concept and methodology of protein microarrays was first introduced and illustrated in antibody microarrays in 1983 in a scientific publication and a series of patents. The high-throughput technology behind the protein microarray was relatively easy to develop since it is based on the technology developed for DNA microarrays, which have become the most widely used microarrays.

Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure.

DNA fragmentation is the separation or breaking of DNA strands into pieces. It can be done intentionally by laboratory personnel or by cells, or can occur spontaneously. Spontaneous or accidental DNA fragmentation is fragmentation that gradually accumulates in a cell. It can be measured by e.g. the Comet assay or by the TUNEL assay.

Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins. Computational methods typically use computer programs to analyze proteins. However, many experimental methods require computational analysis of the raw data.

<span class="mw-page-title-main">Plasmid preparation</span> Biological method of DNA extraction and purification

A plasmid preparation is a method of DNA extraction and purification for plasmid DNA. It is an important step in many molecular biology experiments and is essential for the successful use of plasmids in research and biotechnology. Many methods have been developed to purify plasmid DNA from bacteria. During the purification procedure, the plasmid DNA is often separated from contaminating proteins and genomic DNA.

<span class="mw-page-title-main">Forensic biology</span> Forensic application of the study of biology

Forensic biology is the application of biological principles and techniques in the investigation of criminal and civil cases. Forensic biology is primarily concerned with analyzing biological and serological evidence in order to obtain a DNA profile, which aids law enforcement in the identification of potential suspects or unidentified remains. This field encompasses various sub-branches, including forensic anthropology, forensic entomology, forensic odontology, forensic pathology, and forensic toxicology.

<span class="mw-page-title-main">Semen analysis</span> Scientific analysis of semen

A semen analysis, also called seminogram or spermiogram, evaluates certain characteristics of a male's semen and the sperm contained therein. It is done to help evaluate male fertility, whether for those seeking pregnancy or verifying the success of vasectomy. Depending on the measurement method, just a few characteristics may be evaluated or many characteristics may be evaluated. Collection techniques and precise measurement method may influence results. The assay is also referred to as ejaculate analysis, human sperm assay (HSA), sperm function test, and sperm assay.

RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA. Several methods are used in molecular biology to isolate RNA from samples, the most common of these is guanidinium thiocyanate-phenol-chloroform extraction. The filter paper based lysis and elution method features high throughput capacity.

<span class="mw-page-title-main">Spin column-based nucleic acid purification</span> Method of purifying nucleic acids

Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions.

Cell sorting is the process through which a particular cell type is separated from others contained in a sample on the basis of its physical or biological properties, such as size, morphological parameters, viability and both extracellular and intracellular protein expression. The homogeneous cell population obtained after sorting can be used for a variety of applications including research, diagnosis, and therapy.

Rape investigation is the procedure to gather facts about a suspected rape, including forensic identification of a perpetrator, type of rape and other details.

Forensic serology is the detection, identification, classification, and study of various bodily fluids such as blood, semen, saliva, and urine, and their relationship to a crime scene. A forensic serologist may also be involved in DNA analysis and bloodstain pattern analysis. Serology testing begins with presumptive tests which gives the analyst an indication that a specific bodily fluid may be present, but cannot completely confirm its presence. Following the presumptive tests, confirmatory tests are done on the same sample to confirm what the unknown substance actually is.

<span class="mw-page-title-main">Forensic DNA analysis</span> Genetic analyses in crime analysis

DNA profiling is the determination of a DNA profile for legal and investigative purposes. DNA analysis methods have changed countless times over the years as technology changes and allows for more information to be determined with less starting material. Modern DNA analysis is based on the statistical calculation of the rarity of the produced profile within a population.

References

  1. Horsman KM, Barker SLR, Ferrance JP, Forrest KA, Koen KA, Landers JP. Separation of Sperm and Epithelial Cells in a Microfabricated Device: Potential Application to Forensic Analysis of Sexual Assault Evidence. Anal. Chem. 77, 742 749 (2005).
  2. Greenspoon SA, Ban JD, Sykes K, Ballard EJ, Edler SS, Baisden M, Covington BL. Application of the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System to the extraction of forensic casework samples. J. Forensic Sci. 49, 29-39 (2004).
  3. Voorhees JC, Ferrance JP, Landers JP. Enhanced elution of sperm from cotton swabs via enzymatic digestion for rape kit analysis. J. Foren. Sci. 51, 574-579 (2006).
  4. Nature Methods “Differential lysis in a single-tube extraction process for accurate forensic profiling”, 8 May 2012
  5. Standard Operating Procedure of the differential extraction using a single-tube approach