Microorganism Genomic Profil e Genome : The total genes content of microorganism on the entire DNA strands (nuclear or extra nuclear (Plasmid), Mitochondrium.

Slides:



Advertisements
Similar presentations
Recombinant DNA Technology
Advertisements

Recombinant DNA technology
BCM208 Metabolic Biochemistry Topic 7: Gene metabolism and Expression.
Bacterial Transformation
Recombinant DNA Introduction to Recombinant DNA technology
Recombinant DNA Technology
Gene Cloning Techniques for gene cloning enable scientists to prepare multiple identical copies of gene-sized pieces of DNA. Most methods for cloning pieces.
Manipulating the Genome: DNA Cloning and Analysis 20.1 – 20.3 Lesson 4.8.
DNA Technology and Genomics. Recombinant DNA n Definition: DNA in which genes from 2 different sources are linked n Genetic engineering: direct manipulation.
TOOLS OF GENETIC ENGINEERING
CHAPTER 17 Recombinant DNA and Biotechnology
Chapter 20: Biotechnology. Essential Knowledge u 3.a.1 – DNA, and in some cases RNA, is the primary source of heritable information (20.1 & 20.2)
DNA Technology- Cloning, Libraries, and PCR 17 November, 2003 Text Chapter 20.
Chapter 20~DNA Technology & Genomics. Who am I? Recombinant DNA n Def: DNA in which genes from 2 different sources are linked n Genetic engineering:
AP Biology Ch. 20 Biotechnology.
Chapter 9 – DNA-Based Information Technologies
Trends in Biotechnology
-The methods section of the course covers chapters 21 and 22, not chapters 20 and 21 -Paper discussion on Tuesday - assignment due at the start of class.
歐亞書局 PRINCIPLES OF BIOCHEMISTRY Chapter 9 DNA-Based Information Technologies.
Chapter 20 DNA Technology. DNA Cloning  Gene cloning allows scientists to work with small sections of DNA (single genes) in isolation. –Exactly what.
Molecular Biology (MLMB-201) Lecturer: Dr. Mohamed Salah El-Din Department of Medical Laboratory Technology Faculty of Allied Medical Science.
1 Genetics Faculty of Agriculture Instructor: Dr. Jihad Abdallah Topic 13:Recombinant DNA Technology.
DNA Technology Chapter 20.
Molecular Genetics Techniques BIT 220 Chapter 20.
Ch. 20 Biotechnology. DNA cloning yields multiple copies of a gene or other DNA segment Gene cloning and other techniques, collectively termed DNA technology,
 It is the methods scientist use to study and manipulate DNA.  It made it possible for researchers to genetically alter organisms to give them more.
Section 2 Genetics and Biotechnology DNA Technology
Recombinant Technololgy
Tools of Human Molecular Genetics. ANALYSIS OF INDIVIDUAL DNA AND RNA SEQUENCES Two fundamental obstacles to carrying out their investigations of the.
DNA Technology. Overview DNA technology makes it possible to clone genes for basic research and commercial applications DNA technology is a powerful set.
Biotechnology.
19.1 Techniques of Molecular Genetics Have Revolutionized Biology
PHARMACOBIOTECHNOLOGY.  Recombinant DNA (rDNA) is constructed outside the living cell using enzymes called “restriction enzymes” to cut DNA at specific.
By Melissa Rivera.  GENE CLONING: production of multiple identical copies of DNA  It was developed so scientists could work directly with specific genes.
KEY CONCEPT Biotechnology relies on cutting DNA at specific places.
GENETIC ENGINEERING CHAPTER 20
Chapter 10: Genetic Engineering- A Revolution in Molecular Biology.
Chapter 20: DNA Technology and Genomics - Lots of different techniques - Many used in combination with each other - Uses information from every chapter.
Molecular Biology II Lecture 1 OrR. Restriction Endonuclease (sticky end)
Tools of Molecular Genetics M. Dianatpour PhD
Plasmids that contain l cos sites.
Genetic Engineering/ Recombinant DNA Technology
DNA Technology Ch. 20. The Human Genome The human genome has over 3 billion base pairs 97% does not code for proteins Called “Junk DNA” or “Noncoding.
Chapter 20 DNA Technology and Genomics. Biotechnology is the manipulation of organisms or their components to make useful products. Recombinant DNA is.
Trends in Biotechnology
Plan A Topics? 1.Making a probiotic strain of E.coli that destroys oxalate to help treat kidney stones in collaboration with Dr. Lucent and Dr. VanWert.
RECOMBINANT DNA DNA THAT CONTAINS DNA SEGMENTS OR GENES FROM DIFFERENT SOURCES. DNA TRANSFERRED FROM ONE PART OF A DNA MOLECULE TO ANOTHER, FROM ONE CHROMOSOME.
Recombinant DNA Reverse genetics Synthesis of DNA probes Restriction enzymes, plasmids and recombinant DNA Genomic and cDNA libraries Applications.
Chapter 14 GENETIC TECHNOLOGY. A. Manipulation and Modification of DNA 1. Restriction Enzymes Recognize specific sequences of DNA (usually palindromes)
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Using Restriction Enzymes to Make Recombinant DNA Bacteria and Archaea have evolved.
DNA Technology & Genomics CHAPTER 20. Restriction Enzymes enzymes that cut DNA at specific locations (restriction sites) yielding restriction fragments.
Biotechnology.
Gene Cloning Techniques for gene cloning enable scientists to prepare multiple identical copies of gene-sized pieces of DNA. Most methods for cloning pieces.
Figure 20.0 DNA sequencers DNA Technology.
Chapter 7 Recombinant DNA Technology and Genomics
Bacterial Transformation
Dr. Peter John M.Phil, PhD Assistant Professor Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)
Cloning Overview DNA can be cloned into bacterial plasmids for research or commercial applications. The recombinant plasmids can be used as a source of.
Chapter 20: DNA Technology and Genomics
DNA Technology and Genomics
DNA Technology Now it gets real…..
Material for Quiz 5: Chapter 8
Chapter 20 – DNA Technology and Genomics
Chapter 14 Bioinformatics—the study of a genome
Recombinant DNA Technology
Recombinant DNA Technology
Recombinant DNA Unit 12 Lesson 2.
Chapter 9 Molecular Genetic Techniques and Genomics
Chapter 20: DNA Technology and Genomics
Restriction Enzyme Digestion of DNA
Presentation transcript:

Microorganism Genomic Profil e Genome : The total genes content of microorganism on the entire DNA strands (nuclear or extra nuclear (Plasmid), Mitochondrium. Gene study: structure and location (anatomy) and function (physiology). Value of the gene study: 1- Identification and diagnosis. 2- Typing, classification and taxonomy. Screening Procedures: 1- Species specific primers : Primer is a short chain (around 20 bases bp) of DNA strand from 5’ to 3’. 2-Screening to isolate one particular clone from a gene library involves using a nucleic acid probe for hybridization. The probe will bind to the complementary sequence allowing the required clone to be identified. 3-Restriction enzymes. 4- Sequencing. 5- Bioinformatic.

Primers

PCR3-Agarose gel electrophoresis The final productUV visualisation 3-4 hours

Probe

1-In Situ Methods of Molecular Diagnosis: Hybridization HPV L gene in skin cells Fluorescence in situ hybridization (FISH) analysis 2-Line Probe 3-Microarray: hubdreds of oligonucleotides idetection within few millimeters

19/04/ Restriction Endonucleases Enable to cut the DNA chain for cloning and gene extraction. These restriction enzymes help in protecting bacteria from viruses as they cut the viral DNA leading to stop its work. While the bacteria protect its DNA from these enzymes by methylation enzymes. Each restriction enzyme cut at specific site on the DNA sequence (specificity). Stagger or Sticky ends. Restricion enzymes are divided according to the nature of cutting into two groups: 1-cut the DNA chain straight leading to blunt ends. 2- cut at irregular pattern leading to Stagger or Sticky ends.

19/04/ Palindrome, Restriction Enzyme, Sticky Ends GAATTC G AATTC G Sticky Ends (Cohesive Ends) EcoRI CIVIC, Madam Juang RH (2004) BCbasics 5’….ACTGTACGAT ATCGCTA….3’ 3’….TGACATGCTA TAGCGAT….5’ Straight, Blunt Ends…. EcoRV

Methods of Molecular Diagnosis: Restriction (PFGE, pulse field gel electrophoresis. RFLP, random fragment length polymorphism) M M PFGE profile of Sma I DNA digests of eight VRE isolates from hospital patients RFLP analysis of adenovirus isolates using Hind III

19/04/ DNA Ligase: DNA ligase enzymes: ligating two ends of DNA strand by rebinding phosphodiester found between 5’ PO 4 and 3’OH bonds. RFLP's MapRFLP's Map : A map used to show areas of DNA cuts by using different restriction enzymes which help to study and identify certain areas on this DNA strand. 5’….ACTGTACAGATCCGCTA….3’ 3’….TGACATGTCTAGGCGAT….5’

DNA Sequencing The two main methods of DNA sequencing are the Maxam and Gilbert chemical method in which end-labeled DNA is subjected to base-specific cleavage reactions prior to gel separation, and Sanger's enzmic method. The latter uses dideoxynucleotides as chain terminators to produce a ladder of molecules generated by polymerase extension of a primer. RNA Sequencing: A set of four RNases that cleave 3 ׳ to specific nucleotides to produce a ladder of fragments from end- labeled RNA, using polacrylamide gel electrophoresis (PAGE) analysis allowing the sequence to be read. Sequence Databases: Newly determined DNA, RNA and protein sequences are entered into databases (EMBL and GenBank). These collections of all known sequence for the presence of patterns (eg. Restriction enzyme sites) or similarities (eg. To new strain sequences).

Genome Sequencing Projects: The entire genome sequences of several organisms have been determined (viruses, bacteria, yeast worm and fly) and those of other organisms ( plant, mouse and human) are in progress. Often a genetic map is first produced to aid the project. Genomics: The study of organism's genome concerns with the number, location, overall size and organization of all the genes needed to make up an organism. The position of pseudo-genes will aid our understanding of genome evolution. The immediate challenge is to try to discover the function of the huge numbers of unknown genes predicted by genome sequencing projects. This will require large scale gene inactivation method i.e functional genomics, coupled with proteomic approach. Further advances in automated DNA sequencing may use DNA chips which are high density arrays of different DNA sequences on a solid support as glass or nylon.

Methods of Molecular Diagnosis: Sequencing

Sequencing of PCR-Amplified Segment of the UL97 CMV Gene to Show Point Mutations

DNA Libraries Libraries made from genomic DNA are called genomic libraries and those made from complementary DNA are known as cDNA libraries. The latter lack nontranscribed genomic sequences (repetitive sequences,etc) Good gene libraries are representative of the starting material and have not lost certain sequences due to cloning artifacts. Size of Library: A gene library must contain a certain number of recombinants for a high probability of it containing any particular sequence. This value can be calculated if the genome size and the average size of the insert in the vector are known. Genomic DNA: For making libraries, genomic DNA usually prepared by protease digestion and phase extraction is fragmented randomly by physical shearing or restriction enzyme digestion to give a size range appropriate for the chosen vector. Often combinations of restriction enzymes are used to partially digest the DNA. Vectors: Plasmids, λ phage, cosmid, BAC or yeast artificial chromosome vectors can be used to construct genomic libraries. The choice depending on the genome size. The upper size limit of these vectors is about 10, 23, 45, 350 and 1000 kb respectively. The genomic DNA fragments are ligated to the prepared vector molecules using T4 DNA ligase.

DNA Libraries cDNA Libraries: Genomic libraries are easier to make and contain all the genome sequence. cDNA libraries using prokaryotic mRNA is useless since it is very unstable in the other hand cDNA libraries using eukaryotic mRNA is very useful because the cDNA have no introns sequences and can thus be used to express the encoded protein in E. coli. Since they are derived from mRNA cDNA represent the transcribed parts of the genome (i.e the gene rather than the nontranscribed DNA) furthermore each cell type or tissue expresses a characteristic set of genes (which may alter after stimulation or during development). mRNA preparation from particular tissues usually contain some specific sequences at higher abundance eg. Globin mRNA in erythrocytes. Bioinformatics: Analysis of biological data by computer using special software programs such as NCBI (National Center for Biological Information) in which sequence alignment is adopted making use of DNA libraries.

Saudi Central Asia Africa Indonesia China SE Asia USA West East Worldwide DNA Libraries

Phylogenetic analysis