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 Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercellu ...

and

sperm cells A spermatozoon (; also spelled spermatozoön; ; ) is a motile sperm cell, or moving form of the haploid cell that is the male gamete. A spermatozoon joins an ovum to form a zygote. (A zygote is a single cell, with a complete set of chromosomes, ...

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. Greenspoon et al. 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. Recently a multistep

nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main ...

extraction procedure was introduced to provide DNA lysates of highest quality. 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

Differential lysis in a single-tube extraction process for accurate forensic profiling

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.Standard Operating Procedure of the differential extraction using a single-tube approach
/ref> The first part of the differential

lysis Lysis ( ) is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic (that is, "lytic" ) mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a ''lysate''. In molecular b ...

is carried out under mild conditions to gain the female DNA.

Lysate Lysis ( ) is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic (that is, "lytic" ) mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a ''lysate''. In molecular bi ...

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 An autosome is any chromosome that is not a sex chromosome. The members of an autosome pair in a diploid cell have the same morphology, unlike those in allosomal (sex chromosome) pairs, which may have different structures. The DNA in autosom ...

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.

References

DNA profiling techniques