Unit-IV GENETIC ENGINEERING Introduction to Genetic Engineering Slide 2 Genetic Engineering The study of genes, how genes produce characteristics, and how the characteristics are inherited is called as Genetics. Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. New DNA may be inserted in the host genome by first isolating and copying the genetic material. Slide 3 Significance of Genetic Engineering An organism that is generated through genetic engineering is considered to be a genetically modified organism (GMO). The first GMOs were bacteria in 1973; GM mice were generated in 1974. Insulin-producing bacteria were commercialized in 1982 and Genetically modified food has been sold since 1994. Genetic engineering techniques have been applied in numerous fields including research, agriculture, industrial biotechnology, and medicine. Enzymes used in laundry detergent and medicines such as insulin and human growth hormone are now manufactured in GM cells, experimental GM cell lines and GM animals such as mice or zebrafish are being used for research purposes, and genetically modified crops have been commercialized. zebrafish Slide 4 DNA Fingerprinting It is a technique that identifies individuals on the basis of short pieces of DNA. As no two people have the same nucleotide sequences, they do not generate the same lengths of DNA fragments when their DNA is cut with enzymes. Slide 5 Slide 6 DNA Fingerprinting Technique DNA profiling (also called DNA testing, DNA typing, or genetic fingerprinting) is a technique employed by forensic scientists to assist in the identification of individuals by their respective DNA profiles.DNA DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier. DNA profiling should not be confused with full genome sequencing. It is used in, for example, parental testing and criminal investigation. Slide 7 Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different to distinguish one individual from another, unless they are monozygotic twins. DNA profiling uses repetitive ("repeat") sequences that are highly variable, called variable number tandem repeats (VNTRs), particularly short tandem repeats (STRs). VNTR loci are very similar between closely related humans, but so variable that unrelated individuals are extremely unlikely to have the same VNTRs. Slide 8 DNA fingerprinting in Plant samples Slide 9 Process of DNA fingerprinting It includes the following basic stages: 1.DNA is obtained from a source, which may be as small as one cell. 2.Polymerase Chain Reaction PCR is used to make copies of portions of DNA that contains VNTRs. 3.Restriction enzymes are used to cut the VNTRs DNA into pieces so that VNTRs can be detected. 4.The pieces are separated by Electrophoresis. 5.Comparisons between patterns can be made. Slide 10 The DNA profiling technique was first reported in 1984 by Sir Alec Jeffreys at the University of Leicester in England, and is now the basis of several national DNA databases.national DNA databases Dr. Jeffreys's genetic fingerprinting was made commercially available in 1987, when a chemical company, Imperial Chemical Industries (ICI), started a blood-testing centre in England. Slide 11 DNA Fingerprinting Applications In criminal investigations, the DNA fingerprint of a suspect's blood or other body material is compared to that of the evidence from the crime scene to see how closely they match. The technique can also be used to establish paternity. First developed in the mid-1980s, DNA fingerprinting has been accepted in most courts in the United States. DNA fingerprinting is generally regarded as a reliable forensic tool when properly done, but some scientists have called for wider sampling of human DNA to insure that the segments analyzed are indeed highly variable for all ethnic and racial groups. Slide 12 The techniques used in DNA fingerprinting also have applications in paleontology, archaeology, various fields of biology, and medical diagnostics. In biological classification, it can help to show evolutionary change and relationships on the molecular level, and it has the advantage of being able to be used even when only very small samples, such as tiny pieces of preserved tissue from extinct animals, are available. Slide 13 Polymerase Chain Reaction-PCR Polymerase Chain reaction is a laboratory procedure for copying selected segments of DNA from larger DNA molecules. With PCR, a single cell can provide enough DNA for analysis and identification. Scientists start with a sample of DNA that contains the desired DNA region. Slide 14 Targeting specific portions of DNA for replication enables biochemists to manipulates DNA more easily. When more copies of DNA have been produced it is easy to find, recognize, and manipulate. PCR is a test-tube version of the cellular DNA replication process and require similar components. The DNA from the sample specimen serves as the template for replication. Slide 15 A) During one cycle of PCR, the template DNA is denatured, so that the two strands of DNA separate. This allows the primers to attach to the template DNA. B) DNA polymerase create DNA by extending the primers. C) The same process occurs again, but the previous round of replication has made more template available for further replication. Each subsequent cycle essentially doubles the amount of DNA. Slide 16 Slide 17 Electrophoresis It is a technique used to separate molecules, such as nucleic acids, proteins, or carbohydrates. It separates the nucleic acids on the basis of size. DNA is too long for scientists to work with when taken directly from the cell. To make the DNA more manageable, scientists cut the DNA into smaller pieces with the help of restriction enzymes. Now, electrophoresis is used to separate different sized DNA fragments. Slide 18 1. Electrophoresis uses an electric current to move DNA through a gel matrix. DNA has a negative charge due to phosphate that link the nucleotides. DNA migrates towards the positive pole. The speed at which DNA moves depends on the length of the DNA molecule. Longer DNA molecules moves slowly than the shorter DNA molecules. Slide 19 Basic Review 1. DNA finger printing- Directly examines nucleotide sequence. Examines segments of DNA, which vary in lengths between individuals. Transfer DNA from one person to another. Uses stem cells. Slide 20 2. A technique that separates DNA fragments of different lengths is- Electrophoresis. DNA sequenecing PCR DNA fingerprinting. Slide 21 3. Restriction enzymes- cut DNA randomly. Cut DNA as specific sequences. Can create sticky ends. Both b and c are correct. Slide 22 1. Explain DNA fingerprinting with the help of a diagram. 2. What are the steps of PCR technique? 3.Enlist the applications of DNA fingerprinting. 4.Why can DNA in an organism be used to make the same protein in another organism?