We think you have liked this presentation. If you wish to download it, please recommend it to your friends in any social system. Share buttons are a little bit lower. Thank you!
Presentation is loading. Please wait.
Published byEden Crewse
Modified about 1 year ago
©2000 Timothy G. Standish Hybridization And Southern Blots Timothy G. Standish, Ph. D.
©2000 Timothy G. Standish Hybridization The bases in DNA will only pair in very specific ways: G with C and A with T In short DNA sequences, imprecise base pairing will not be tolerated Long sequences can tolerate some mispairing only if hydrogen bonding of the majority of bases in a sequence exceeds the energy required to overcome mispaired bases The source of any single strand of DNA is irrelevant, merely the sequence is important, thus complimentary DNA from different sources can form a double helix This phenomenon of base pairing of single stranded DNA strands to form a double helix is called hybridization as it may be used to make hybrid DNA composed of strands from different sources
©2000 Timothy G. Standish Hybridization DNA from source “Y” TACTCGACAGGCTAG CTGATGGTCATGAGCTGTCCGATCGATCAT DNA from source “X” TACTCGACAGGCTAG Hybridization
©2000 Timothy G. Standish Hybridization Because DNA sequences will seek out and hybridize with other sequences with which they base pair in a specific way much information can be gained about unknown DNA using single stranded DNA of known sequence Short sequences of single stranded DNA can be used as “probes” to detect the presence of their complimentary sequence in any number of applications including: –Southern blots –Northern blots (in which RNA is probed) –In situ hybridization –Dot blots... In addition, the renaturation, or hybridization, of DNA in solution can tell much about the nature of organism’s genomes
©2000 Timothy G. Standish Library Screening The most common method of library screening involves hybridization of probes to target DNA Hybridization refers to the specific way DNA sequences base pair with their exact compliment Probes - Single stranded nucleic acids used to hybridize with a target DNA. Generally probes are radioactive or marked in some other way so that they can easily be identified after binding to target DNA To design probes for hybridization screening, something must be known in advance about the target sequence
©2000 Timothy G. Standish Hybridization Screening Takes advantage of the fact that complimentary strands of DNA can recognize one another By sticking DNA from many colonies or plaques in a library to a membrane Making the DNA single stranded Then hybridizing a probe to the DNA on the membrane thus marking target DNA on the membrane, colonies or plaques containing the target DNA can be identified
©2000 Timothy G. Standish Cover with X-ray film Develop X-ray film Hybridization Screening Membrane Transfer cells to membrane Lyse cells - DNA and protein stick to membrane Locate colony with target clone Block membrane - Prevents probe from sticking to membrane Add probeWash off excess probe
©2000 Timothy G. Standish Southern Blots Called Southern blots after their inventor Involve four steps: 1Digestion of DNA using restriction enzymes 2Separation of the DNA fragments by size using gel electrophoresis 3Transfer of fragments to a nitrocellulose or nylon membrane 4Hybridization of a probe to the fragment or fragments of interest 5Probe detection (autorad development)
©2000 Timothy G. Standish Making A Southern Blot Digestion and Electrophoresis Experimental 3 Marker 1 Control 2
©2000 Timothy G. Standish Membrane Making A Southern Blot 3 DNA Transfer To Membrane DNA Gel Buffer Gel Membrane Paper Towels
©2000 Timothy G. Standish Membrane with bound DNA Addition of blocking reagent Parts of the membrane not already covered with DNA now bind blocking reagent Probe covers the membrane, but only binds to complimentary DNA Probe addition After washing Probe only remains annealed to complimentary DNA Making A Southern Blot 4 Probe Hybridization
©2000 Timothy G. Standish Fragments complimentary to the probe appear as bands on the autorad Making A Southern Blot 5 Autorad Development Membrane with probe bound to complimentary DNA X-ray film is placed over the membrane and left until radiation from the probe has exposed the film
©2000 Timothy G. Standish So What is the Big Deal? Southern blots tell both the size and something about the sequence of a fragment mixed in with many other fragments, thus they can be used for many purposes RFLPs - Restriction Fragment Length Polymorphisms - When mutations change the size of specific sequences in the genome, they may be identified by a change in the size of a RE digested fragment identified using a Southern blot RFLPs - Can be correlated to specific genetic defects thus allowing diagnosis of genetic disease RFLPS - Are used as genetic markers in mapping genes Some parts of DNA called Variable Number Tandem Repeats (VNTRs) are highly polymorphic (variable) in length and can be used for genetic fingerprinting
©2000 Timothy G. Standish Using DNA Fingerprinting A murder victim is found to have human tissue under his fingernails having, during the course of struggle, managed to scratch his murderer Two suspects have been identified as having a motive and suspicious scratches on their faces: Suspect 1 - The victims wife Suspect 2 - The wife’s boyfriend Under Nails Suspect 1 Victim Suspect 2 A VNTR Probe was used to make this DNA fingerprint
©2000 Timothy G. Standish Does This Prove The Boyfriend Did It? Not exactly DNA fingerprinting is very good for elimination of suspects. Thus the wife’s tissue was not under her husbands fingernails That the tissue under the husbands fingernails was the murderer’s is an assumption There are other plausible scenarios including - The husband and boyfriend were fighting, the wife came home and shot her husband to protect her boyfriend The boyfriend’s DNA matches tissue under the victims nails, while this eliminates millions of other people it does not prove it is actually his tissue
©2000 Timothy G. Standish DNA Fingerprinting Can Show Relationships During the 1970s and 80s a “dirty war” was fought in Chile directed by General Pinochet Many people disappeared and their children were adopted by innocent people These children have been reunited with their grandparents using DNA fingerprinting Maternal Grandmother Paternal Grandmother Father Paternal Grandfather Maternal Grandfather Child Mother
©2000 Timothy G. Standish
DNA fingerprinting. DNA fingerprinting is used to determine paternity Look at the DNA of the mother, father and child Could these parents produce this.
Genetic fingerprinting Each DNA profile is unique!
LEQ: HOW DOES DNA PROFILING WORK? 12.8 to NUCLEIC ACID PROBES Short single strands of DNA w/ specific nucleotide sequences are created using.
DNA Technology Chapter 20. Plasmid Use Plasmids are good tools for DNA Technology Can be isolated from bacterial cells Often carry resistance genes Isolated.
Chapter 5 Nucleic Acid Hybridization Assays A. Preparation of nucleic acid probes: 1. Labeling DNA & RNA - Nick Translation - Random primed DNA labeling.
Amanda Barrera Biology Honors Period 1 DNA Sequencing is the process of determining the order of base pairs in a section of DNA.
HOW MANY CATs? A DNA Profiling Simulation. Structure of DNA Double helix (Analogy: Spiral staircase) Anti-parallel strands 4 Bases (A, C, T, and G) Complementary.
Chapter 20 DNA Technology and Genomics. Biotechnology is the manipulation of organisms or their components to make useful products. Recombinant DNA is.
Kinship DNA Fingerprinting Simulation Grab the packet from the front table and begin reading.
Spawned naming of related techniques: Southern blot (DNA) Northern blot (RNA) Western blot (Protein) Eastern blot (???)
Analyzing DNA Fragments AP Biology Fall DNA Fingerprint DNA fingerprint: unique array of base sequences in each organism that is slightly different.
Advanced Molecular Biological Techniques. Polymerase Chain Reaction animation.
Lecture 3 – Selection of Recombinants & clone analysis The white colonies will all be recombinants, but only one of these many colonies will contain the.
DNA. A. Terminology A. Terminology Chromosomes- strands of genetic material Chromosomes- strands of genetic material Genes- Fundamental unit of heredity.
Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik.
1 Northern & Southern Hybridization Avalon Garcia
Biotech. Cloning a mammal PCR This is the polymerase chain reaction. It is a technique to multiply a sample of DNA many times in a short period of time.
DNA profile analysis is based on the use of the “Southern” hybridization technique to analyze the polymorphic regions of human DNA, which are the regions.
Forensic Science DNA Analysis 1. History of Biological Evidence in Forensics DNA fingerprinting Also known as DNA profiling Used with a high degree.
1 Chapter 5: Hybridisation & applications Hybridisation Southern In situ RFLP micro-array & SNP analysis.
DNA fingerprinting Every human carries a unique set of genes (except twins!) The order of the base pairs in the sequence of every human varies In a single.
DNA Fingerprinting and Forensic Analysis Chapter 8.
Trends in Biotechnology Constructing and Screening a DNA Library.
©2001 Timothy G. Standish Isaiah 40:4, 5 4Every valley shall be exalted, and every mountain and hill shall be made low: and the crooked shall be made straight,
Revision – Concept map. Human Genome Project Procedures –Genetic mapping –Physical mapping –DNA sequencing Techniques –Use of microsatellites (STRs) –Restriction.
Biotech Continued… How do forensic scientists determine who’s blood has been left at a crime scene? How do forensic scientists determine who’s blood.
Introduction to DNA DNA Fingerprinting and the Polymerase Chain Reaction.
Chapter 17: Variable-Number Tandem Repeats Profiling.
Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa.
Forensic Science: Fundamentals & Investigations, Chapter 7 1 Chapter 7 DNA Fingerprinting By the end of this chapter you will be able to: explain how crime.
Module 1 Section 1.3 DNA Technology AS Biology. Specification.
DNA Fingerprinting. Also known as DNA profiling Used in criminal and legal cases since the 1980’s to determine identity or parentage Also used to identify.
DNA Fingerprinting & Forensic Analysis. How is DNA Typing Performed? Only one-tenth of 1% of DNA differs in each person; this variation can create.
Biotech. Southern Blotting Through a series of steps, DNA that has been separated by electrophoresis is applied to a membrane of nylon or nitrocellulose.
© SSER Ltd.. Genetic fingerprinting is a technique that was developed in 1984 by Alec Jeffreys and his colleagues at the University of Leicester The human.
Lecture 11: Variable-Number Tandem Repeats Profiling.
DNA Profiling. There are two methods used to carry out DNA profiling There are two methods used to carry out DNA profiling DNA profiling using a probe.
Manipulation of DNA. Restriction enzymes are used to cut DNA into smaller fragments. Different restriction enzymes recognize and cut different DNA sequences.
PCR Polymerase chain reaction. PCR is a method of amplifying (=copy) a target sequence of DNA.
RFLP Analysis Restriction Fragment Length Polymorphism 1.Extract/Isolate DNA from the cell restriction enzymes 2.Cut DNA into smaller fragments using restriction.
AP Biology Chapter 20. Biotechnology: DNA Technology & Genomics.
Chapter 10: Genetic Engineering- A Revolution in Molecular Biology.
Genetic screening. What the spec says about “medical diagnosis” The use of labelled DNA probes and DNA hybridisation to locate specific genes. Once located,
SOUTHERN BLOTTING Submitted To: Submitted By: Mr. Harsh Vishal Sehgal Lecturer B.Tech – Biotech.
How do you identify and clone a gene of interest? Shotgun approach? Is there a better way?
Homework Prepare for 6x task 2 and 6x written assessment June 2012 June 2011 June 2010 Bring marked corrected papers to tues 23 rd april Test DNA technol.
©2001 Timothy G. Standish Romans 5:17 17For if by one man’s offence death reigned by one; much more they which receive abundance of grace and of the gift.
Human Genomics. Writing in RED indicates the SQA outcomes. Writing in BLACK explains these outcomes in depth.
DNA and Gene Cloning -DNA revision -Gene cloning -cDNA and reverse transcriptase -Restriction endonucleases.
© 2017 SlidePlayer.com Inc. All rights reserved.