Molecular Probes Mitesh Shrestha
Molecular Probes Small DNA segments (genomic DNA, cdna or synthetic oligonucleotides) or RNA segments (often synthesized on DNA template) that recognize complementary sequences in DNA or RNA molecules and thus allow identification and isolation of these specific DNA sequences from an organism. Antibodies are also occasionally use as probes to recognize specific protein sequences. Used for a variety of other purposes including diagnosis of infectious diseases, identification of food contaminants, variety of microbiological tests, forensic tests (e.g. fingerprinting of murderers or rapists), etc. Used to identify strains of an organism e.g. varieties of a crop species (a plant breeder likes to have quick test to identify his variety to maintain his patent or breeder's right, so that a competitor may not use it in another name leading to infringement of rights).
Molecular Probes DNA/RNA probe assays are faster and sensitive, so that many conventional diagnostic tests for viruses and bacteria involving culturing of the organism, are being fast replaced by antibody and DNA probe assays. While culture tests can take days or even months, molecular probe assays can be performed within few hours or minutes. Therefore, the use of DNA probes has become today's most sophisticated and sensitive technology for a variety of uses involving biological systems both in basic and applied studies including their commercial use.
Nucleic Acid Hybridization Nucleic acid hybridization is a fundamental tool in molecular genetics which takes advantage of the ability of individual singlestranded nucleic acid molecules to form double stranded molecules (that is, to hybridize to each other)
Application of Molecular Probes Evolutionary Studies Molecular Cytogenetics Applications in Medical Research Detection of Pathogenic Microorganisms Detection of Changes to Nucleic Acid Sequences Detection of Tandem Repeat Sequences (DNA Fingerprinting)
What is DNA Fingerprinting? In 1985 an English geneticist, Alec Jeffreys, was studying inherited variation within genes and among individuals. He studied a genetic peculiarity known as the intron. Introns are sequences of junk DNA that do not code for a specific protein. He noticed that some introns are made up of the same DNA base pair sequences and that the number of repetitions varies from person to person.
The different sequence segments that vary in size and composition and have no apparent function are called minisatellites The different sequences is the same as the word "POST" has a different meaning from "STOP" or "POTS," even though they use the same letters. i
DNA Fingerprinting Using these sequences, every person could be identified solely by the sequence of their base pairs There are so many millions of base pairs, the task would be very timeconsuming Instead, scientists are able to use a shorter method, because of repeating patterns in DNA. These patterns do not, however, give an individual "fingerprint," They are able to determine whether two DNA samples are from the same person, related people, or non-related people.
DNA Fingerprinting using VNTR's On some human chromosomes, a short sequence of DNA has been repeated a number of times. the repeat number may vary from one to thirty repeats these repeat regions are usually bounded by specific restriction enzyme sites cut out the segment of the chromosome containing this variable number of tandem repeats (VNTR's ) identify the VNTR's for the DNA sequence of the repeat.
Making DNA Fingerprints DNA fingerprinting is a laboratory procedure that requires six steps: 1: Isolation of DNA. 2: Cutting, sizing, and sorting. Special enzymes called restriction enzymes are used to cut the DNA at specific places
3: Transfer of DNA to nylon. The distribution of DNA pieces is transferred to a nylon sheet by placing the sheet on the gel and soaking them overnight. 4-5: Probing. Adding radioactive or colored probes to the nylon sheet produces a pattern called the DNA fingerprint.
4-6: DNA fingerprint. The final DNA fingerprint is built by using several probes (5-10 or more) simultaneously.
Practical Applications of DNA Fingerprinting 1. Paternity and Maternity person inherits his or her VNTRs from his or her parents Parent-child VNTR pattern analysis has been used to solve standard father-identification cases
2. Criminal Identification and Forensics DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared FBI and police labs around the U.S. have begun to use DNA fingerprints to link suspects to biological evidence blood or semen stains, hair, or items of clothing
3. Personal Identification The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed
4. Diagnosis of Inherited Disorders Diagnose inherited disorders in both prenatal and newborn babies These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia, and many others.
Considerations when evaluating DNA evidence In the early days of the use of genetic fingerprinting as criminal evidence, given a match that had a 1 in 5 million probability of occurring by chance the lawyer would argue that this meant that in a country of say 60 million people there were 12 people who would also match the profile.
2. Problems with Determining Probability A. Population Genetics VNTRs, because they are results of genetic inheritance it will vary depending on an individual's genetic background
B. Technical Difficulties Errors in the hybridization and probing process must also be figured into the probability Until recently, the standards for determining DNA fingerprinting matches, and for laboratory security and accuracy which would minimize error
When evaluating a DNA match, the following questions should be asked: -Could it be an accidental random match? -If not, could the DNA sample have been planted? -If not, did the accused leave the DNA sample at the exact time of the crime? -If yes, does that mean that the accused is guilty of the crime?
Assignment Write various methods of probe labeling. [7.5] Write the NBT/BCIP reaction.[2.5] Write the advantages and disadvantages of Radio active labeling. [2.5] Write the advantages and disadvantages of Non - Radio active labeling. [2.5] Write about DNA fingerprinting. [7.5]