Short Tandem Repeat (STR) Analysis

STR analysis works to examine individual areas in DNA. The differences from the collective areas of one person to another can allow for distinguishing between individuals. In criminal investigations, there are thirteen regions that are analysed and compared to establish profiles. In fact, DNA databases used at the government level involve the sequence of these thirteen regions. The chances of two people having the exact same thirteen regions is virtually impossible - likely one in a billion. A common DNA joke is that a person's odds of winning the lottery are higher than finding a perfect match for the thirteen regions.

Y-Chromosome Analysis

Since the Y chromosome passes from a male to his son, analysing genetic markers on a Y chromosome can be of aid in identifying familial ties in males or for analysing any evidence entailing many males. Another benefit of Y-chromosome analysis is to establish a family line over many generations.

There are other types of analysis but these are some of the main traditional and current methods used to analyse DNA. No doubt, new techniques will be developed that will be even more rapid, successful and cost-effective.

Mitochondrial DNA Analysis

Mitochondrial DNA analysis works well on samples that are unable to be analysed through RFLP or STR analysis. There are two kinds of DNA in the cell - mitochondrial DNA and nuclear DNA. With other types of analysis, nuclear DNA is removed from the sample but with mitochondrial DNA analysis, DNA is removed from the cell's mitochondria. Sometimes, a sample can be old and will no longer have nuclear material in the cell, which poses a problem for the other types of DNA analysis. With mitochondrial DNA analysis, however, mitochondrial DNA can be removed, thus having important ramifications for cases that were not solved over many years. This means that mitochondrial DNA analysis can be very valuable in investigations for a missing person. Mitochondrial DNA will be the same from a woman to her daughter because it is passed on from the egg cell.

Recombinant DNA and genetic techniques

Recombinant DNA (or rDNA) is made by combining DNA from two or more sources. In practice, the process often involves combining the DNA of different organisms. The process depends on the ability to cut and re-join DNA molecules at points which are identified by specific sequences of nucleotide bases called restriction sites. DNA fragments are cut out of their normal position in the chromosome using restriction enzymes (also called restriction endonucleases) and then inserted into other chromosomes or DNA molecules using enzymes called ligases.

Gene Cloning

This describes the process of copying fragments of DNA which can then be used for many different purposes, such as creating GM crops, or finding a cure for disease.  There are two types of gene cloning:  in vivo, which involves the use of restriction enzymes and ligases using vectors and cloning the fragments into host cells (as can be seen in the image above).  The other type is in vitro which is using the polymerase chain reaction (PCR) method to create copies of fragments of DNA.

For in vivo cloning a fragment of DNA, containing a single gene or a number of genes, is inserted into a vector that can be amplified within another host cell. A vector is a section of DNA that can incorporate another DNA fragment without losing the capacity for self-replication, and a vector containing an additional DNA fragment is known as a hybrid vector. If the fragment of DNA includes one or more genes the process is referred to as gene cloning.

Cloning DNA in Plasmids

Contributor: Genome Management Information System, Oak Ridge National Laboratory, U.S. Department of Energy Genome Programs  http://genomics.energy.gov

There are 4 different type of vectors:

• Plasmid vectors
• Lamda (λ) phage vectors
• Cosmids
• Expression vectors

The host cell copies the cloned DNA using its own replication mechanisms. A variety of cell types are used as hosts, including bacteria, yeast cells and mammalian cells.

Polymerase Chain Reaction (PCR)

Source:  Andy Vierstraete 

http://users.ugent.be/~avierstr/principles/pcr.html

This is an in vitro method for making many copies of a specific section of DNA, without the need for vectors or host cells.  The DNA to be copied – the template DNA – is mixed with forward and reverse primers complementary to the end of the template DNA, nucleotides, and a version of DNA polymerase known as Taq polymerase. (This enzyme is stable under high temperatures, and is obtained from the thermophilic bacterium Thermus aquaticus.) The process involves the repetition of three steps:

• denaturation, which separates the two nucleotide strands of the DNA molecule
• primer annealing, in which the primers bind to the single-stranded DNA
• extension, in which nucleotides are added to the primers – in the 5' to 3' direction – to form a double-stranded copy of the target DNA.

Each cycle takes a few minutes, and repeated cycles can produce large amounts of a specific DNA sequence in a matter of hours rather than days. However, this cloning method does require knowledge of some details about the nucleotide sequence to be copied, and the technique is very sensitive to small amounts of contamination.

Gene Libraries

Source: http://www.emunix.emich.edu/~rwinning/genetics/index.htm

A gene library is a large collection of cloned DNA sequences from a single genome.  A genomic library, (as can be seen above) in theory, would contain at least one copy of every sequence in an organism’s genome. These are used to investigate the structure of a given chromosome, or to clone specific genes.  These types of libraries may be prepared from a subset of the entire genome (for example, a single chromosome). The first step in creating a genomic library is to break up, or ‘fractionate’, the genome using physical methods or restriction enzymes. The fragments are then linked to appropriate vectors and cloned in a suitable host cell population.

A cDNA library (complementary DNA) contains DNA present in a given cell population which is prepared from the mRNA (messenger RNA) using the enzyme reverse transcriptase.  The resulting cDNA represents the genes expressed in the cell population as a subset of the entire genome, and can be cloned using a vector and suitable host cell (as seen in the diagram above). The cDNA will not include introns or regulatory sequences as these are removed from the RNA during processing, and this makes a cDNA library easier to maintain.  A cDNA library can also be prepared using reverse transcriptase PCR (RT-PCR).

The Identification of Gene Products in a Gene Library

Restriction enzymes (to cut the DNA) and gel electrophoresis (to separate the resulting fragments) can be used to produce a physical map of DNA segments in a process known asrestriction mapping.  An example of what one of these may look like can be seen below.

Source: University of Leicester

There are also a number of techniques that can be used to identify specific genes or gene products within a gene library and these are: Southern blotting, Northern blotting andWestern blotting. However, the most powerful experimental technique for investigating genetics at the molecular level is DNA sequencing, which allows the nucleotide sequences of genes – even whole chromosomes – to be determined. Automated sequencing technologies are now allowing us to sequence the entire genomes of organisms from bacteria to human beings.

Molecular Genetics and Biotechnology

The new techniques of molecular genetics, combined with developments in associated biotechnologies, have led to advances in a number of different fields. We can now analyse the genomes of species that make an important contribution to agriculture, fuel production or drug development. We can move specific genes from one organism to another to createtransgenic plants and animals, and use animal cloning techniques to produce animals that are genetically identical, such as Dolly the sheep, and more recently, cloned pets such as cats and dogs.

The process of cloning is straightforward, but the results are not always predictable.  It took many hundreds of attempts to get it to work and produce one live sheep, and cloning in itself raises many questions not only about benefits and risks but also many ethical questions.

Diagram of pigs to show how animal cloning is carried out.  Source: National Human Genome Research Institute

The technique of genetic fingerprinting, which enables the identification of individuals and the relationships between individuals has found many applications in science today. There is also ongoing research into gene therapy which examines the possibility of introducing cloned genes to compensate for defective, mutant genes. And other areas, for example,human cloning and stem cell research open up many ethical issues that must be addressed alongside the scientific developments.

The Value of DNA Evidence

DNA is a powerful investigative tool because, with the exception of identical twins, no two people have the same DNA. In other words, the sequence or order of the DNA building blocks is different in particular regions of the cell, making each person's DNA unique. Therefore, DNA evidence collected from a crime scene can link a suspect to a crime or eliminate one from suspicion in the same way that fingerprints are used. DNA also can identify a victim through the DNA of relatives if a victim's body cannot be found. For example, if technicians have a biological sample from the victim, such as a bloodstain left at a crime scene, the DNA taken from that evidence can be compared with DNA from the victim's biological relatives to determine if the bloodstain belongs to the victim. When a DNA profile developed from evidence at one crime scene is compared with a DNA profile developed from evidence found at another crime scene, they can be linked to each other or to the same perpetrator, whether the crime was committed locally or in another state.

DNA evidence in the form of saliva, blood, skin tissue, hair, and semen are often recovered from crime scenes and can be crucial to the investigation of sexual assaults and other violent crimes. For example, during a sexual assault, biological evidence such as hair, skin tissue, semen, blood, or saliva can be left on the victim's body or at the crime scene. In addition, hair and fiber from clothing, carpet, bedding, or furniture could be transferred to the victim's body during an assault. This evidence is helpful in proving that there was physical contact between an assailant and a victim. DNA properly collected from the victim, crime scene, or suspect can be compared with known samples to place the suspect at the scene of the crime. If there is no suspect, however, a DNA profile of the crime scene can be entered into the Federal Bureau of Investigation's (FBI) Combined DNA Index System (CODIS), which allows agencies to match DNA profiles with other profiles entered into local, state, and national databases to identify a suspect or link serial crimes.

As with fingerprints, the effective use of DNA as evidence may require the collection and analysis of elimination samples to determine whether biological evidence came from a suspect or someone else. When investigating sexual assault or rape cases, it may be necessary to obtain an elimination sample, such as a blood or saliva sample, from the victim's relatives or consensual sex partner to account for all of the DNA found on the victim or at the crime scene.

Case Studies: The Power of a DNA Match

Nothing illustrates the power of DNA evidence more effectively than the case studies–or real–life experiences-of those whose lives have been changed by such evidence. Whereas some case studies demonstrate DNA's ability to exonerate inmates wrongfully convicted of crimes, others show the powerful sense of closure and relief that a DNA match can bring to victims of violent crime. The three very different case studies presented below reflect the power of a DNA match and reveal some of the complexities involved in the criminal justice system. Given the pain suffered and the time irrevocably lost, these individuals' stories also indicate an urgent need to improve the capabilities and response times of DNA databases and eliminate the growing backlog of rape kits.

A Lifetime Struggle: The Courage of Kellie Greene

Kellie Greene's life changed forever late one January evening more than 7 years ago following a visit to the laundry room in her apartment complex. As she opened the door to her apartment, she was brutally attacked by an intruder who smashed a tea kettle over her head and then raped her. At some point during the vicious attack, which lasted 45 minutes, Kellie's rapist used dishwashing detergent. It is unknown whether the rapist used it as a lubricant, after ejaculation to cleanse himself, or purposely to destroy crucial DNA evidence that ultimately could convict him of the assault. In any case, forensic experts with the Florida Department of Law Enforcement were able to retrieve a sample of the rapist's semen from the sweater Kellie wore that night. It was this key DNA evidence that, on February 28, 1997, linked David William Shaw to Kellie's attack on January 18, 1994. More than a month would pass, however, before she was told of the DNA match in April 1997.

The road to recovery for Kellie, and countless other rape survivors, is paved with anger, loss, rage, sadness, numbness, confusion, shame, guilt, fear, despair, and courage. The rape is a memory that never disappears and one that marks a woman's life forever. The experience shapes how she reacts to life's challenges and unexpected turns, how she gets through each day, how she sleeps at night, how she feels about her sexuality, how she feels about her body, and how she feels about men. "I think I always will struggle with the sexuality. It's never the same. Something that should be natural becomes something that you have to work at," Kellie said.

After Kellie's brutal attack and rape, she did not hesitate to report it to the authorities. "There wasn't any question. I was beat up really badly," she said. But once at the hospital, Kellie had to wait 3 hours in a hospital bed with her head wound still bleeding because the hospital would not treat her without first being seen by a medical examiner. It took seven staples to close the gash in her head.

At the time of her rape, Florida was not processing nonsuspect cases because of funding issues, and, as a result, DNA evidence in her case sat on a shelf for more than 3 years before it was analyzed. If it had not been for persistent law enforcement officers, particularly one detective, Kellie's rape kit might still be sitting on a shelf. Because officers thought Kellie's rape was similar to rapes occurring in Daytona Beach, less than 2 hours north of Orlando where Kellie's attack occurred, her rape kit was dusted off and examined. Once the results were entered into Florida's local DNA database, a hit was made via the FBI's CODIS system, allowing for an almost immediate match. Her rapist's DNA profile did not match the profile of the rapist in Daytona Beach but that of a man already serving a 25–year sentence for beating and raping a woman 6 weeks before attacking Kellie.

While Kellie's rapist remains behind bars today, she continues to fight to keep him there. Quirks in the criminal justice system, insensitivity toward the victim, and human error allowed her case to slip through the cracks more than once, resulting in a significantly reduced sentence for the offender. Not until late April 2000 was Kellie informed of a plea agreement stating that Shaw could serve concurrently a 22-year sentence for Kellie's rape, a 15-year sentence for a robbery, a 5-year sentence for obstructing justice, and the 25-year sentence for the first rape. A motion filed by the defense attorney to clarify the sentence never reached the state's attorney's office. Finally, the judge signed orders denying Kellie restitution and denying her request that Shaw be treated with chemical castration shots. As a result, Kellie's rapist could be released from jail as early as 2001. Had consecutive sentences been ordered for his brutal crimes, he would not be released until 2041.

After her trial, Kellie drafted and introduced a bill in the Florida legislature that would mandate consecutive sentences for convicted sex offenders and murderers in prison who are found guilty of subsequent offenses. Sponsored by Representative Randy Johnson (R), the legislation was called the Sexual Predator Prosecution Act of 2000. The bill passed Florida's House and Senate unanimously and was signed into law in June 2000.

Kellie has been speaking out about her rape and recovery for more than 6 years. In October 1999, she formed a nonprofit organization named SOAR-Speaking Out About Rape, Inc. She travels across the country giving rape awareness seminars about the healing process and the importance of DNA evidence in solving cases. SOAR gave her recovery a purpose. "I was able to learn something from it and to help others. So often people think of the rape only and not the aftereffects, she pointed out. "DNA is really an amazing tool. You don't know where you're going to get the DNA from but you can get it from a lot of places."

A First Step Toward Healing: Crime Victim Debbie Smith's Story

Everything changed for rape victim Debbie Smith when the man who had raped her 6 years earlier was identified. When processed through Virginia's DNA databank, the DNA sample of her assailant collected years earlier had produced a match or "hit" with DNA of an inmate in a Virginia prison. As reflected by her compelling testimony before the National Institute of Justice's National Commission on the Future of DNA Evidence, that DNA match gave Debbie final proof that her assailant would not "come back" for her, as he had threatened. What is more important is that it allowed her to begin healing.

Debbie's ordeal began at about 1 p.m. on May 3, 1989, at her home in Williamsburg, Virginia. She was cleaning house, doing laundry, and baking a cake. A light rain was falling, and her husband–a police lieutenant–was upstairs sleeping after working the night shift and appearing in court that morning. After stepping outside briefly, Debbie came back in and, for some reason, left the door unlocked. Within a few minutes, a masked stranger entered Debbie's house and nearly destroyed her life. The stranger dragged Debbie to a wooded area. He blindfolded her. He robbed her. And he raped her repeatedly, telling her, "Remember, I know where you live and I will come back if you tell anyone."

When allowed to return home, Debbie told her husband about the attack but in fear begged him not to call the police. She just wanted to take a shower and wash away the pain. Debbie's husband, however, convinced her to notify the police and visit a hospital where trained medical personnel could examine her and collect physical evidence that might identify the rapist. If she showered, that evidence would be lost. Debbie thanks God every day for her husband's advice. Although she was "plucked and scraped and swabbed" during her visit to the hospital, Debbie's rape examination kit produced the crucial DNA 

evidence that ultimately identified her attacker.

True peace of mind came for Debbie Smith on July 26, 1995, when a forensic scientist for the Commonwealth of Virginia notified Debbie that a DNA match had been made. Her assailant was serving time in a Virginia prison for a separate offense. For the first time since the rape, Debbie knew that her attacker could not come after her. Debbie learned later that her assailant had gone to jail only months after raping her. Because of a backlog in Virginia's DNA database, she waited 6 years to hear about it.

Proof of Innocence: Inmate Ronald Cotton's Story

Ronald Cotton's story begins on a summer night in 1984 when two rapes were committed in Burlington, North Carolina. In each case, an assailant entered an apartment, cut the phone wires, raped a woman at knifepoint, and stole money and other items. Both victims were taken to the hospital, where full rape examination kits were completed.

The first victim, 22-year-old Jennifer Thompson, described her attacker as a tall African-American man in his early 20s. Police collected photographs of area men meeting that description, including 22-year-old Ronald Cotton, a Burlington resident employed at a restaurant near Thompson's apartment. Cotton had two prior convictions: one for breaking and entering, and another for assault with intent to rape. Thompson selected Cotton from police photos as her rapist. When Cotton visited the police station to clear up the misunderstanding, he only strengthened the case mounting against him. He claimed that he had been with friends on the night of the rapes, but those friends did not corroborate his alibi. At a physical lineup of suspects, Thompson again selected Cotton. In August 1984, police arrested Cotton and took him into custody. In January 1985, Cotton was convicted of Thompson's rape and sentenced to life in prison. That verdict, however, was overturned, and a new trial was ordered. Cotton was optimistic given a crucial discovery he had made about one of his fellow inmates, Bobby Poole–a tall African–American young man from Burlington also convicted of rape who bore a strong resemblance to the composite sketch used in Cotton's case. Poole had reportedly bragged to inmates that he had committed the rapes for which Cotton was serving time.

The second trial was even more devastating than the first. Both victims testified against Cotton; the jury did not believe that Poole was the real assailant; and, most damaging of all, the court withheld evidence of Poole's alleged confessions. Convicted of both rapes, Cotton received two life sentences plus 55 years in prison.

Back in prison, Cotton "waited it out" for years. In 1994, however, he learned about DNA testing (a procedure unavailable at the time of his trials). He filed and won a motion for DNA testing. In 1995, Burlington police turned over to the court all case evidence containing semen or other bodily fluids. Samples from Jennifer Thompson had deteriorated and could not be tested, but those from the second victim provided a breakthrough for Cotton. On a tiny vaginal swab, scientists found a bit of sperm. Subjected to PCR testing, that sample showed no match to Ronald Cotton. He could not have committed the crime.

The state DNA database matched the sample to Bobby Poole. On June 30, 1995, almost 11 years after the rapes and 10 1/2 years after being taken into custody, Ronald Cotton was cleared of all charges and released from prison.

Microarray-based DNA decoding

A DNA microarray is a device for high-throughput investigations widely used in molecular biology and in medicine. It consists of an arrayed series of microscopic spots (‘features’ or ‘locations’) containing few picomoles of oligonucleotidescarrying a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes tohybridize a DNA or RNA sample under suitable conditions. Probe-target hybridization is usually detected and quantified byfluorescence-based detection of fluorophore-labeled targets to determine relative abundance of the target nucleic acidsequences. Microarray has been used for the successfully decoding of ESAC DNA-encoded libraries. The codingoligonucleotides representing the individual chemical compounds in the library, are spotted and chemically linked onto themicroarray slides, using a BioChip Arrayer robot. Subsequently, the oligonucleotide tags of the binding compounds isolated from the selection are PCR amplified using a fluorescent primer and hybridized onto the DNA-microarray slide. Afterwards,microarrays are analyzed using a laser scan and spot intensities detected and quantified. The enrichment of the preferential binding compounds is revealed comparing the spots intensity of the DNA-microarray slide before and after selection.