The Use of Electrophoresis in Forensic Science

Fig. 1 - Gel Electrophoresis of DNA

Electrophoresis has proved to be an invaluable tool in the analysis of crime scene evidence, especially in the area of DNA fingerprinting.

Electrophoresis involves the separation of chemicals along a solid medium in the presence of an applied potential difference. In electrophoresis, chemicals such as blood proteins, DNA or inorganic ions can be separated according to differences in their mass and/or charge. The solid medium used in electrophoresis is usually an agarose or polyacrylamide gel

Electrophoretic separation has uses in forensic science because it can be used to isolate and compare DNA, blood proteins and inorganic substances such as gunshot residues from crime scenes with suspects, victims or standard reference material.

Blood Protein Analysis Using Gel Electrophoresis

This process involves the separation of ‘marker’ proteins that are found on the surface of red blood cells. Many of these are antigens that determine particular blood groupings such as A, B, AB and O, and they can therefore be used to exclude suspects from being present at a crime scene.

Blood protein analysis has now been largely replaced by DNA fingerprinting of blood, because this latter method is much more specific. It can still serve a role as collaborative evidence, however, and has also had great relevance in past criminal cases.

DNA Analysis Using Gel and Capillary Electrophoresis

Fig. 2 - Capillary Electrophoresis

Electrophoresis is most frequently used in forensic science to produce DNA fingerprints, as illustrated in figure 1. DNA evidence from a crime scene can be compared to DNA samples from different suspects, for instance, and suspects can either be included or excluded from suspicion using the results of such tests

In gel electrophoresis, DNA strands from crime scenes, victims or suspects are applied to an agarose gel that is subjected to an electric potential. The more traditional RFLP (restriction fragment length polymorphism) profiling procedure is now being replaced by the PCR (polymerase chain reaction) method, which often involves the use of shorter DNA segments known as STRs (single tandem repeats). This method is faster and requires less DNA.

Capillary electrophoresis (figure 2), in which a fused silica capillary is used instead of a gel slab, is now being used more frequently in DNA electrophoresis. Although applying the same principles of separation as the more traditional gel slab electrophoresis, it is more rapid and has a higher resolution.

Inorganic Ion Analysis Using Capillary Electrophoresis

Capillary electrophoresis can also be used to separate and analyse anions found in explosives and poisons so that the substances used in crimes can be identified and even linked to suspects.

Anions capable of being isolated from explosive residues include azides, chlorates, chlorides, nitrates, nitrites, perchlorates, sulfates and thiocyanates, while the anions of interest in toxic chemicals are azides, cyanide, arsenates, arsenites, chromates, thiosulfates, oxalates, bromides and iodides.

Capillary electrophoresis is often also used in conjunction with ion chromatography to achieve more effective separation of ions.

Criminal Cases Where Electrophoresis Has Been Employed

Blood protein analysis was used as recently as the O.J. Simpson trial in 1995, when electrophoretic comparisons between O.J’s blood and blood from the crime scene showed they both had the factors A, ESD1 and PGM2+2-. This evidence was highly incriminating, as the probability of two samples having all these factor is just 0.44%.

O.J’s guilt was also suggested by DNA tests carried out on his blood, which revealed the probability of his being innocent was 1 in 57 billion. The main reason he wasn’t convicted was due to the seed of doubt the defence sowed in the minds of the jurors about possible interference with the evidence by the FBI.

Using the many DNA databases now available, such as CODIS, for instance, DNA samples from a crime scene can be compared with the DNA from suspects and victims related to that crime. Chester Dewayne Turner , for instance, was linked by DNA evidence to deaths in the U.S. as far back as March, 1987.

About 25% of violent crime cases in the U.S. since 1989, however, have in fact resulted in the exoneration of suspects because of DNA profiling procedures. For example, after spending 21 years in an Indiana prison for rape, DNA tests now indicate Larry Mayes was innocent. He was subsequently released in 2002. By 1996, over 108 post-conviction exonerations had in fact occurred in the USA using DNA profiling.

The anions of inorganic salts and acids are often found in gunshot and explosive residues, and also in foodstuffs that have been adulterated with poisons. Capillary electrophoresis can rapidly profile and often quantify these chemicals, and this method therefore has merit as collaborative evidence in forensic science.

Future Directions in Electrophoresis

Research is currently being undertaken in the US to develop portable microchip DNA profiling devices that can be used in the field. In this method, STR analysis of a small DNA sample can be achieved on the surface of microchips in much less time than traditional techniques. ‘Pulsed field electrophoresis’ is another innovation being investigated – here, the direction of the electric field is alternated, allowing for the separation of DNA molecules up to several million base pairs in length.

These, and other advances in electrophoretic technology, will ensure faster and more effective analysis of crime scene evidence in the years ahead.

References

Petricevic, S., 2010, "DNA Profiling in Forensic Science", nzic.org.nz, accessed 1/2/2010

Saferstein, R., 2004, "Criminalistics, an Introduction to Forensic Science", Pearson Education, New Jersey

Tissue, B.M, 2010, "Electrophoresis", The Chemistry Hypermedia Project, chem vt.edu, accessed 1/2/2010

How Successful Was Copenhagen?

How Successful Was Copenhagen?

The Copenhagen climate conference would arguably have been a total failure if it had not been for the 'Copenhagen Accord' initiated by President Obama.

The Copenhagen Accord was brokered by Barack Obama, Wen Jiabao, the Chinese premier. along with 25 other nations. Although most parties to this accord agreed that global temperatures could not rise above 2 degrees Celsius, no firm resolution to reduce emissions was achieved. The accord was effectively one of intent only and is not legally binding. Indeed, parties to the accord merely agreed to ‘take note’ of it.

The Road to Copenhagen

Initially, Copenhagen was intended to lay the basis for a legally binding carbon reduction scheme beyond 2012, when the conditions of the 1997 Kyoto Protocol are due to expire. Alternatively labelled ‘COP 15’, this conference was the 15th Conference of Parties set up by the United Nations Framework Convention on Climate Change (UNFCCC).COP 11 in Montreal, COP 12 in Nairobi, COP 13 in Bali and COP 14 in Poland involved negotiations leading up to Copenhagen, and much hope was held for the outcomes of COP 15 in these gatherings.

The ‘Bali Road Map’, for instance, which emerged from COP13, included plans to reduce deforestation, and to negotiate issues of mitigation, adaptation, technology and financing among UNFCCC countries. All nations present signed the agreement to consider these issues. Results in Poland were less optimistic; several developed nations, including Japan and Australia, rejected a further decrease in mid-term reduction goals and there was no general consensus among industrialised nations on long term emission targets.

COP14 did, however, produce practical technological and financial emission reduction strategies that could be adopted by developing countries. It was envisaged that Copenhagen would cement these goals into some sort of enforceable extension of Kyoto’s achievements.

What Were the Achievements of Kyoto?

To understand the Copenhagen agreement it must then be viewed in the light of what was achieved in Kyoto. The 1997 Kyoto Protocol involved a legally binding agreement among the 186 countries that have ratified it. It stipulates that the 39 ‘Annex 1’, or developed, countries reduce their collective greenhouse gas emissions by 5.2% (compared to 1990 levels) by 2012.

Means of achieving these reductions are permitted to be flexible and range from emissions trading schemes, ‘Joint Implementation’ (in which developed countries receive ‘emission reduction units’ for assisting transitional countries to reduce their emissions) and the ‘Clean Development Mechanism’, where developed countries receive credits for financing emission reduction projects in poorer nations.

Non Annex 1 countries- the so-called ‘developing nations’, were given no legally binding emission reduction targets but were given incentives to develop carbon reduction projects in exchange for carbon credits.

Enforcement of the Kyoto Protocol

The above reductions are enforceable as a result of the Marrakesh Accords, which developed a compliance system for Kyoto. Delegates from the Kyoto enforcement committee are permitted to enforce sanctions against Annex 1 countries that do not meet their emission targets or who fail to regularly submit emission updates. Countries that exceed their emissions targets will be required to make up the difference as soon as possible in addition to reducing emissions by another 30%. They will also be temporarily excluded from emissions trading schemes.

But how enforceable are these emission targets in reality? Ulfstein and Werksman point out that some of the chosen deterrents in the Kyoto protocol, such as increasing emission targets by 30% for infringing parties, may not be as effective as has been predicted. Moreover, Halvorssen and Hovi have stated that ‘ A country that deliberately fails to abide by other legally binding commitments under the Kyoto Protocol is also likely to resist the application of punitive consequences, regardless of whether these consequences are made legally binding or not.’

Outcomes of Copenhagen

In the light of this, the fact that Copenhagen achieved nothing legally binding may not be of consequence. What may be more important is the involvement of the United States, previously notorious for its non ratification of Kyoto, and the intentions agreed to. Some of these include the establishment of the ‘Copenhagen Green Carbon Fund’, which pledges 30 million dollars in the next three years, with a planned 100 billion per year by 2020 to vulnerable developing nations to help mitigate and adapt to the effects of climate change.

Another major consensus was that global temperatures should not increase by more than 2 degrees, and that the Accord should be reviewed by 2015 to discuss whether 1.5 degrees would be a better target. In addition, developing nations have agreed to submit reports of their efforts to reduce greenhouse gas emission every two years.

Nevertheless, many parties to the conference have left dissatisfied, and for various reasons. Vulnerable low lying nations such as the Cook Islands, Barbados and Fiji wanted no less than a legally binding agreement for a 1.5 degree increase in global temperatures signed both by Annex 1 and developing nations. India and China, on the other hand, wanted to continue the conditions of Kyoto so that they would remain, as non Annex 1 countries, excepted from binding emissions targets. The African group of nations walked out of talks at one stage because they could not see India, China and the U.S., all major emitters, agreeing to any binding targets.

Perhaps, then, the world needs to wait to see what the consequences of the Copenhagen Accord will be. This may happen as soon as the climate talks in Mexico later this year.

References

Copenhagen Climate Council, 2009, ‘What is the Kyoto Protocol’?, copenhagenclimatecouncil.com

Halvorssen, H., and Hovi, J., 2006,’The Nature, Origin and Impact of Legally Binding Consequences: the Case of the Climate Regime’, Springer Netherlands,Springerlink.com , accessed 6/2/2010

The German Marshall Fund of the United States, 2009, ‘Clinging to Kyoto’,gmfus.org, accessed 7/2/2010

Ulfstein, G and Werksman, J., The Kyoto Compliance System: Towards Hard Enforcement, folk.uio.no, accessed 8/2/2010

Vidal, Stratton, Goldenberg, 2009,’Low targets, goals dropped: Copenhagen ends in failure’, guardian.co.uk, accessed 6/6/2010

Monoclonal Antibodies

Fig. 1 - Mouse Cholera Antibody

Monoclonal antibodies are showing enormous potential as biological tools in the diagnosis and treatment of human disease.

Monoclonal antibodies (MAbs) are immunoglobulin proteins made to target potentially any molecule capable of instigating the production of antibodies. Such molecules are known as antigens. The antigen of concern is injected into mice, where white blood cells known as B cells produce antibodies against it (see figure 1).

The antibody -producing B cells are then hybridised with myeloma tumour cells to form ‘hybridoma’ cells, which multiply quickly, producing clones of themselves and, in turn, vast amounts of antibodies. The term ‘monoclonal’ is derived from the fact that they are produced from one type of cell- the hybridoma cell. This revolutionary procedure was developed by Kohler and Milstein, who were awarded a Nobel Prize for their work in 1984.

Because antibodies are specific for their target antigen only, MAbs have the potential to be more efficient than other drugs, which may also attack normal body cells. As a result, side effects of therapy such as nausea or allergic reactions are reduced.

Monoclonal Antibodies in Human Therapy

Monoclonal antibodies have several uses; the most obvious one being to fight the specific antigen they were produced from. One such example is Mylotarg, a drug derived from a MAb which binds to CD33, a cell-surface molecule expressed by the cancerous cells in acute myelogenous leukemia . Other MAbs approved by the FDA attack tumor cells in lymphomas and some breast cancers, while some target receptor cells on the cancerous white blood cells in chronic lymphocytic leukemia.

The immune system can also be suppressed using monoclonal antibodies; one example is Muromonab-CD3, which binds to the CD3 molecule on the surface of T cells. This acts to prevent the acute rejection of organs after transplant operations.

Despite the moderate success of these and other monoclonal treatments, progress in human therapy has been relatively slow because the human immune system naturally rejects antibodies that have originated in mice. As a result, the antibodies are usually comparatively short lived and have limited success rates. Additional drawbacks can include side effects such as vomiting and fever.

Newer therapies are consequently employing genetically engineered antibodies that combine the antigen binding properties of the mouse antibody with genetic material from human antibodies. This technique aims to reduce rejection to a manageable level. Examples of FDA approved human or chimeric MAbs include Synagis, Herceptin and Remicade,used in the treatment of breast cancer, leukaemia and rheumatoid arthritis respectively.

Monoclonals as Diagnostic Tools

The specificity of MAbs for the antigen that stimulates their production also makes them useful in the detection of these antigens in the body. This has been utilised in the identification of ABO blood groups in human serum and in the diagnosis of pregnancy-related hormones such as HCG in pregnancy testing kits.

Other diagnostic tests include the detection of drug levels, infectious diseases such as AIDS (using the ELISA test), tumour antigens and specific hormones in the human body. While many of these tests employ the use of immunoassay procedures, which quantify the formation of antigen antibody complex (Ag-Ab complex), others involve the attachment of MAbs to radioactive atoms in a process known as radioimmunodetection. In some situations fluorescent molecules or metal atoms such as copper and gold (see figure 2) may also be coupled to the antibody to assist in imaging the target.

Ethical Issues associated with MAbs

The use of mice to produce MAbs has been controversial because of the painful and debilitating effects of the procedure. In order to achieve an enhanced inflammatory immune response in the mouse, for instance, substances called adjuvants are used. These release the antigen into the mouse over a long period of time, which often results in painful lesions at the site of injection. Freund's Complete Adjuvant (FCA) has actually been banned in the Netherlands and the United Kingdom.

This, and the fact that when the required immune response has been achieved, the mouse’s spleen is removed to provide a source of antibody producing cells, has resulted in some European countries legislating to limit MAb production in mice. Alternatives being investigated include the use of tissue culture and DNA technology to produce the antibodies in vitro.

The Future of Monoclonal Antibodies

With anti-rejection technology improving continually, monoclonal antibodies may soon be extended to the therapeutic treatment of diseases outside the traditional areas of oncology, autoimmune and inflammatory disorders, such as infectious diseases and opthamology. With sales exceeding 32 billion dollars in 2008, MAbs are set to become an important sector of the pharmaceutical industry.

References

Kimball, J., 2010, `"Monoclonal Antibodies," Kimball’s Biology Pages, jkimball.ma.ultranet, accessed 23/2/2010

Lynette A. Hart, 1996, "Monoclonal antibodies," Mouse in Science,ucdavis.edu, accessed 23/2/2010

"Uses of Monoclonal Antibodies," Molecular-Plant-Biotechnology.info, accessed 25/2/2010

Washington, D.C.: Biotechnology Industry Organization, 1989, "Monoclonal Antibody Technology - The Basics," accessexcellence.org, accessed 25/2/2010