DNA TECHNOLOGY

POLYMERASE CHAIN REACTION Polymerase Chain Reaction (PCR) is used to copy and amplify tiny quantities of DNA. When researchers want to study a particular sequence of DNA, they need many (identical) copies of it. The traditional method of cloning (using plasmids) takes a lot of time and work.

POLYMERASE CHAIN REACTION Instead, a “photocopier for DNA” is used. It uses enzymes to replicate DNA. PCR is carried out in a thermal cycler. The process was developed in 1983 by Kary Mullis who received a Nobel Prize for his work.

PCR – the Process The desired DNA is heated, which breaks the hydrogen bonds between the strands. Primers are added to start the process of DNA replication. As the mixture is cooled, the primers bond to the original (but now single) strands. Nucleotides and a thermostable DNA polymerase is added. These bond with the “exposed” bases and the DNA polymerase joins them. Each strand is now complementary.

PCR – the Process These new strands are reheated and function as templates for the next round. The process continues. The DNA is exponentially amplified.

PCR

PCR Demo

Gel Electrophoresis Gel electrophoresis is a technique used to separate fragments of DNA. Gel electrophoresis is a technique used to separate fragments of DNA. Separates fragments based on size. Separates fragments based on size. Most types use Agarose to separate fragments. Most types use Agarose to separate fragments. Agarose is a porous gel. It can allow the passage of molecules through, however, larger molecules move more slowly through it since they cannot squeeze through the pores as easily as smaller molecules. Agarose is a porous gel. It can allow the passage of molecules through, however, larger molecules move more slowly through it since they cannot squeeze through the pores as easily as smaller molecules. Electrophoresis Apparatus

ELECTROPHORESIS TECHNIQUE An agarose gel is casted with several holes called wells at one end. An agarose gel is casted with several holes called wells at one end. The gel is placed in an electrophoresis box which is filled with an electrolyte buffer solution. The gel is placed in an electrophoresis box which is filled with an electrolyte buffer solution. Samples of DNA are placed in the wells. Samples of DNA are placed in the wells.

ELECTROPHORESIS TECHNIQUE Electrical leads are attached to the ends of the box creating an electrical potential across the apparatus. Electrical leads are attached to the ends of the box creating an electrical potential across the apparatus. Because DNA has a negative electrical charge. It is "pulled" towards the positive side of the apparatus. Because DNA has a negative electrical charge. It is "pulled" towards the positive side of the apparatus.

ELECTROPHORESIS TECHNIQUE The smaller molecules travel faster through the agarose. The smaller molecules travel faster through the agarose. Over time this separates the various sized fragments of DNA. Over time this separates the various sized fragments of DNA. The gel is then removed and stained for DNA. This results in a gel which shows several bands of stained DNA. The gel is then removed and stained for DNA. This results in a gel which shows several bands of stained DNA.

Finished Gel

GEL ELECTROPHORESIS DEMO Gel Elec. Demo

DNA Fingerprinting DNA is now a powerful tool in identification. DNA is now a powerful tool in identification. Based on the fact that the amount of "junk DNA" differs uniquely between individuals. Based on the fact that the amount of "junk DNA" differs uniquely between individuals. Structural genes are often separated by large regions of repeating basepairs. Structural genes are often separated by large regions of repeating basepairs. The number of these repeats is unique to an individual. The number of these repeats is unique to an individual. Therefore when DNA from a person is cut with a restriction enzyme, the length of the fragments will be unique to an individual. Therefore when DNA from a person is cut with a restriction enzyme, the length of the fragments will be unique to an individual.

DNA Fingerprinting Contd… This will therefore produce a unique banding pattern following a gel electrophoresis. This will therefore produce a unique banding pattern following a gel electrophoresis. This test is highly accurate, and the probability of another individual possessing an identical banding pattern is estimated as around 1:14,000,000,000. This test is highly accurate, and the probability of another individual possessing an identical banding pattern is estimated as around 1:14,000,000,000.

DNA Fingerprinting

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CRIME Forensic scientists use DNA profiles. The chances of two people having exactly the same DNA profile is 30,000 million to 1 (except for identical twins)

BIOLOGICAL MATERIALS USED FOR DNA PROFILING Blood Hair Saliva Semen Body tissue cells

DNA PROFILING CAN SOLVE CRIMES The pattern of the DNA profile is then compared with those of the victim and the suspect. If the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene ( NB:it does not prove they committed the crime). If the profile doesn’t match the suspect then that suspect may be eliminated from the enquiry.

EXAMPLE A violent murder occurred. The forensics team retrieved a blood sample from the crime scene. They prepared DNA profiles of the blood sample, the victim and a suspect as follows:

WAS THE SUSPECT AT THE CRIME SCENE? Suspects Profile Blood sample from crime scene Victims profile

SOLVING MEDICAL PROBLEMS DNA profiles can be used to determine whether a particular person is the parent of a child. A childs paternity (father) and maternity(mother) can be determined. This information can be used in Paternity suits Inheritance cases Immigration cases

EXAMPLE: A PATERNITY TEST By comparing the DNA profile of a mother and her child it is possible to identify DNA fragments in the child which are absent from the mother and must therefore have been inherited from the biological father.

IS THIS MAN THE FATHER OF THE CHILD? MotherChildMan

FAMOUS CASES In 2002 Elizabeth Hurley used DNA profiling to prove that Steve Bing was the father of her child Damien

FAMOUS CASES Colin Pitchfork was the first criminal caught based on DNA fingerprinting evidence. He was arrested in 1986 for the rape and murder of two girls and was sentenced in 1988.

FAMOUS CASES O.J. Simpson was cleared of a double murder charge in 1994 which relied heavily on DNA evidence. This case highlighted lab difficulties.

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