Genetic Engineering

Genetic Engineering 4.4.1

4.4.1 Outline the use polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA.Exploring DNA • In the past few decades astounding genetic techniques have been developed, which enable scientists to explore and manipulate DNA. These include • Copying DNA in a laboratory – the polymerase chain reaction (PCR); • Using DNA to reveal its owner’s identity –DNA profiling; • Mapping DNA by finding every A, T, C and G is – the Human Genome Project. • Cutting and pasting genes to make new organisms – gene transfer. • Cloning cells and animals. • These techniques offer new hope for obtaining treatments and vaccines for diseases; for creating new plants for farmers; for freeing wrongly convicted people from prisons.

Heated Debate • Techniques such as gene transfer and cloning have sparked heated debate. • Is it morally and ethically acceptable to manipulate nature in this way? • Are the big biotech companies investing huge sums of money into this research to help their fellow citizen or are they just in it for economic profit? • With regard to cloning and stem cell research, is it morally and ethically acceptable to create human embryos solely for scientific research?

- • Part of being a responsible citizen is making informed decisions relating to these difficult questions. It is not technical complexity that makes these questions difficult, it is also because humans have never had to face them before.

Polymerase Chain Reaction (PCR) • PCR is a laboratory technique which takes a very small quantity of DNA and copies all the nucleic acids in it to make millions of copies of the DNA. • This is very useful when very small quantities of DNA are found in a sample and larger amounts are needed for analysis. DNA from very small samples of semen, blood or other tissues or even from long-dead specimens can be amplified using PCR. • http://www.youtube.com/watch?v=_YgXcJ4n-kQ

- • PCR is used to solve a very simple problem: how to get enough DNA to be able to analyse it. • Analysis is impossible without the DNA from just one or a few cells. When collecting DNA from the scene of a crime or from a cheek smear, often only a limited number of cells are available. By using PCR, forensics experts or research technicians can obtain millions of copies of the DNA in just a few hours. Such large quantities are large enough to get results from, notably using gel electrophoresis.

PCR PCR was developed in 1983 by KaryMulis who received the Nobel prize for chemistry in 1993 for his work. PCR is carried out in a thermal cycler http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf Good Animation

Steps involved in PCR • PCR is carried out at high temperatures using a DNA polymerase enzyme from Themusaquaticus, a bacterium that lives in hot springs. • This enzyme is able to survive the heating stages of each cycle. • DNA amplification by PCR • In this technique, any specific target segment within a DNA molecule can be quickly amplified (copied many times) in a test tube. Starting with a single DNA molecule, automated PCR can generate 100 billion similar molecules in a few hours. • In principle PCR is simple. A DNA sample is mixed with the DNA replication enzyme DNA polymerase, nucleotide monomers, and a few other ingredients. • The solution is then exposed to cycles of heating (to separate the DNA strands) and cooling.

During each cycle, the DNA is replicated, doubling the amount of DNA. • The key to automating PCR was the discovery of an unusual heat-stable DNA polymerase, first isolated from prokaryotes living in hot springs. This enzyme can withstand the heat at the start of each cycle. • Devised in 1985, PCR has had a major impact on biological research and biotechnology. • PCR has been used to amplify DNA from a wide variety of sources: fragments of DNA from a 40,000-year-old frozen woolly mammoth; DNA from fingerprints or from tiny amounts of blood, tissue, or semen found at crime scenes, DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders, and DNA of viral genes from cells infected with such difficult to detect viruses as HIV.

4.4.2 state that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size • Electrophoresis is a technique used to separate large molecule (nucleic acids or proteins) based on their different rates of movement in an electric field caused by a combination of their charge and their size.

Electrophoresis Gel electrophoresis can separate nucleic acids that differ in size. DNA fragments created by restriction enzymes are separated based on their rate of movement through a gel in an electric field. How far a DNA molecule travels is inversely proportional to its length. After the current is turned off, a dye is added; this reveals the separated bands by fluorescing in ultraviolet light.

http://media.pearsoncmg.com/bc/bc_campbell_biology_7/media/activities/load.html?20&Dhttp://media.pearsoncmg.com/bc/bc_campbell_biology_7/media/activities/load.html?20&D • DNA may be analyzed by gel electrophoresis. In this process DNA molecules migrate to the positive pole as current passes through the gel. • Making the gel is the first step in gel electrophoresis. A common material used for gel electrophoresis of DNA is agarose. Agarose gels are made by first boiling a mixture of powdered agarose and buffer. When the mixture is cooled to about 65oC, the solution is poured into a gel mold. When further cooled to room temperature, the agarose solidifies to produce the gel with indentations called wells.

Steps • The agarose gel is placed in an electrophoresis apparatus and buffer is added to cover the gel. • DNA is loaded into the wells using a micropipettor. Each DNA sample has tracking dyes added to it (usually blue) and either sucrose or glycerol to make the solution dense and "sink" into the well. • After the wells are filled, the power supply is turned on and the DNA moves towards the positive pole. At this stage the DNA band(s) cannot be seen, but the tracking dye (dark blue) allows the progess of the electrophoresis to be followed. The smallest DNA fragments typically follow the blue dye down the gel.

Steps • The DNA is visualized by staining, for example with ethidium bromide, and then destaining in water. The ethidium bromide binds to the DNA and makes it fluoresce orange under UV light. • A photograph of the results is taken and used for analysis and for a permanent record of the experiment.

ElectrophoresisUseful tips • Larger molecules will move more slowly through the gel than smaller molecules. • If a spot of the unknown sample has travelled exactly the same distance as one of the known molecules, they are likely to be the same. • PCR can be used to increase the size of the sample and then gel electrophoresis can be used to compare the sections of DNA with the DNA of various suspects in an investigation

Review and Fun • Watch this animation • http://learn.genetics.utah.edu/content/labs/gel/ • Learn Genetic: Super Flashy Animation

4.4.3 state that gel electrophoresis of DNA is used in DNA profiling • The process of matching an unknown sample of DNA with a know sample to see if they correspond is called DNA profiling. This is also referred to as DNA fingerprinting because of some of the similarities with fingerprints but the techniques are very different. • If, after separation by gel electrophoresis, the pattern of bands formed by two samples of DNA fragments are identical, it means that both most certainly came from the same individual. • If the patterns are similar, it means that the two individuals are probably related.

DNA Profiling • DNA profiling is also known as DNA fingerprinting. It is a technique that compares DNA from different sources without mapping the entire genome since that would take a long time. • DNA profiling is used to compare the DNA of a suspect with, for example, blood found at a crime scene or to compare the DNA of a child with that of an adult who could be the parent. • When a small amount of DNA is found, for example, in a spot of blood on a crime scene, the PCR can be used to increase the amount of DNA. The two strands of DNA double helix are separated. Restriction enzymes or endonucleases, which will cut between specific sequences of organic bases are used to cut the DNA into sections. The sections are separated using gel electrophoresis. • The gel electrophoresis will separate the sections of DNA according to size and charge. This creates a pattern of stripes and bands, determined by the sequence of the organic bases. As every person’s DNA is unique, it would be highly unlikely that two different people would produce the exact same pattern of DNA sections on the gel.

Interpreting DNA Profile • Paternity testing is a form of DNA profiling. The DNA of the man who may be the father of a child is compared to the child's DNA and to that of the mother. • An example of the DNA bands from a paternity test is shown below. The standard band provides a control or comparison with which to compare the samples. • Is the man the father of the child? • a) True b) False

What is DNA Profiling? • Even though each person’s DNA is unique, with current technology it is not practical to look at each difference. Currently in the laboratory we look at 10 different areas of DNA, which are known to vary widely between people. These 10 areas contain short repeating sequences known as Short Tandem Repeats (STR). The number of these repeating sequences varies between individuals. An additional area is also provided which indicates whether the person is male or female. The technique of DNA profiling is centred on analysing and measuring these differences in length.

4.4.4 Describe the application of DNA profiling to determine paternity and also in forensic investigations. • DNA Use in Forensic Cases • Biological fluids such as blood, semen, hair and saliva can play a role in shedding light on various types of criminal cases. In the case of sexual assault cases, semen found on vaginal swabs and /or clothing of a victim can be compared to the DNA profile of a nominated suspect. Similarly in murder/assault cases blood found on the clothing of a nominated suspect can be compared to the DNA profile of the victim. • Contrary to a number of years ago the smallest of traces left behind in criminal offences can now suffice for a DNA analysis. With the currently applied DNA techniques, a profile can be constructed, even of old and (partially) decayed biological traces. • Forensic DNA analysis can also establish the origin of a biological sample with an extremely high degree of probability.

Forensic Investigations • Compare DNA from the suspect with DNA from the crime scene sample • This technique breaks down DNA into sections which are separated by gel electrophoresis. If the DNA bands of a suspect are a match with those found in a sample (e.g. blood, semen, hairs) at the crime scene, then the suspect probably was at the scene although it is not always this sample. • http://en.wikipedia.org/wili/Genetic_fingerprinting • Problems that arise when using DNA profiling as evidence

Paternity Profiling • DNA profiling can be used in paternity suits when the identity of someone’s biological father must be known for legal reasons. DNA profiling to determine paternity uses the concept that all of the child’s DNA comes from its parents. Each band shown on the DNA profile of a a child must correspond with a band in the profile of the father or mother. • DNA technology is used in courts of law • http://in.youtube.com/watch?v=veYl0vigfLk

Use a relative’s DNA to determine the identity of a victim • This technique has also been used to determine the identity of the remains of dead people. People claimed that the Tsar of Russia and his family were shot dead during the Russian Revolution and bodies were shown to prove this. However, not everyone believed that they were really the remains of the Romanovs. The identity of these bodies could not be proven until DNA fingerprinting was brought in. by taking blood samples of relatives of the Romanovs, DNA patterns could be established. Samples from the bodies showed similar DNA patterns and the conclusion was that the bodies were likely to be the Romanov family.

TOK DNA Profiles • Blood typing was used in paternity and forensic testing before the advent of DNA profiling. The outcome of tests using blood typing is so broad that it is only useful in excluding an individual (that is, it can say that the individual dos not have the same blood type as the one searched for. To say that they do have the same blood type would not be useful unless the pool of possible suspects was very small and all with different blood types. • DNA profiling, on the other hand, compares parts of non coding DNA that are unique to each individual (except identical twins). Profiles constructed using appropriate procedures have statistically extremely high reliability rate and as such, are widely used in paternity and forensic cases with great success

4.4.5 Analyse DNA Profiles to draw conclusions about paternity or forensic investigations • Forensic Investigation • A crime has been committed and two suspects are under investigation. DNA profiling has been carried out and the results are shown in the figure (Page No. 66 IB Biology By Minca Peters) it can be seen that the two bands visible in the “sperm DNA” match the DNA bands of suspect 1 but not Suspect 2

Paternity Investigation • When the paternity of a child is investigated, the band found in the child’s DNA are compared to those of the mother and the alleged father. Since all of the child’s DNA comes from its parents, all bands should be found in either parent. Of course, a parent only gives half of its DNA to the child, so not all the parent’s bands can be found in the child • Refer to paternity investigation in the text book IB Biology by Mica and also IB Biology By Heinemann Page number 102 Activity: Page Number 130 IB Biology By C.J Clegg

Genetic Engineering