Genetic Engineering and Biotechnology

  • Biotechnology

    • Use of living organisms to create products that are useful to humans

      • ie vitamins, antibiotics

  • Genetic Engineering

    • Deliberate modification of an organism’s genome sequence

      • crops with desirable charcteristics

  • Recombinant DNA

    • DNA containing fragments from two or more different sources

  • Recombinant DNA Technology

    • Set of procedures used to combine genetic material from multiple sources and create DNA sequences that are otherwise not found in biological organisms

    • Recombinant DNA technologies use procedures to carry out genetic engineering only

  • Standard Cloning Techniques

    • Isolate plasmid from a bacterial cell

    • Isolate the DNA of interst

    • Insert the gene of interest into the plasmid DNA, forming recombinant DNA

    • Insert the recombinant DNA into a bacterium, such as E. Coli

    • Culture the bacteria, growing many identical copies

    • Standard cloning techniques make use of restriction enzymes

  • Restriction Enzymes

    • An endonuclease that is an important tool that allows scientists to cut up DNA at specific sites

    • Naturally produced by bacteria as defense against infection

  • Restriction Sites

    • Short nucleotide sequences between 4 and 8 nucleotides for restriction enzymes

    • Palindromes

      • Symmetrical nucleotides sequences running in opposite directions on two strands

  • Sticky Ends

    • Staggered cut with gaps

    • Add dna from sources

    • DNA ligase seals the strands together

    • recombinant dna

  • Creating Recombinant DNA

    • Requirements

      • Endonuclease (Restriction Enzymes)

      • Cloning Vector

      • Host

  • Techniques to Introduce DNA into a Host cell

    • Electroporation

    • Protoplast fusion

  • Constructing Complementary DNA from RNA

  • Reverse transcriptase

    • turn single stranded RNA into double stranded DNA

    • Synthesizes DNA based on an RNA Template

    • Complementary DNA is DNA produced from an RNA template (cDNA)

    • RNA polymerase is not involved in the process of constructing complementary DNA from RNA

    • RNA polymerase synthesizes RNA not DNA

  • Constructing cDNA

    • Begin with single stranded RNA, apply poly-T primer

    • Add reverse transcriptase and ACGT

    • Add RNaseH to cut up RNA and regenerate primers

    • Add DNA polymerase and DNA ligase to synthesize the new strand

  • DNA molecule can then be inserted into vectors and used in cloning, or amplified using PCR

  • Cloning Vectors

    • Provides means for transferring a gene of interest to a host organism during the cloning process

    • Good cloning vectors

      • ORI

      • Selectable Marker

        • Allowing scientists to easily identify the bacteria with the plasmid that contains the gene of interest

      • Unique Restriction site

        • Polylinkers or Multicloning sites

  • Cloning Vectors

    • Plasmids

      • Self-replicating piece of extrachromosomal DNA found in prokaryotes

        • Most common vector

        • Autonomous

          • Self-replicating

    • Bacteriophage

      • Virus that infects bacteria

    • Cosmid

      • Hybrid plasmid/phage

    • Artificial chromosomes

      • Synthetic chromosomes that contain fragments of DNA integrated into a host chromosome

        • PACs

        • BACs

        • YACs

      • Carry large fragments

      • Humane Genome Project

  • Methods for DNA Analysis

  • Polymerase Chain Reaction (PCR)

    • Quickly and easily making many copies of DNA from any source

      • Priming

      • Thermostable DNA polymerase

        • Taq polymerase

      • Deoxyribonucleotide Triphosphates

        • dATP, dCTP, dGTP, dTTP

      • Thermocycler

        • Cyclically increases and decreases temperature of a solution

  • Two types of PCR

    • End-point PCR

      • Making a large amount of copies

    • Real-time PCR (qPCR)

      • Track how much DNA is in the sample during the reaction

  • Gel electrophoresis

    • Separates nucleic acids and proteins on the basis of their size and electrical charge using a gel

    • DNA and RNA are negatively charged so they migrate toward the positive electrode

  • Blotting Techniques

    • Blotting

      • Combines electrophoresis and hybridization to detect specific fragments

    • Southern Blotting

      • Uses radioactive DNA hybridization probes and autoradiography to identify the presence (or lack thereof) of a specific gene

      • Autoradiography

        • method for detecting radioactively labeled molecules

    • Steps for Blotting

      • Cut the DNA with restriction enzymes

      • Separate the Fragments by size on gel

      • Place a sheet of paper on top of the gel and apply pressure for contact

      • Place the paper in a heat sealed bag with a radioactive probe

      • Wash the filter and place it on xray film to produce an image of the DNA bands

        • Autoradiography

  • Construction of Genomic Libraries

    • Genomic Library

      • Collection of an organism’s entire genomic DNA

      • Involves cleaving the genome and inserting the fragments into vectors

  • Applications of Genetic Engineering

    • Creation of recombinant DNA

      • Scientists can insert DNA from one organism into another organism’s genome through standard cloning

        • Express genes

  • Introducing mutations

    • Site-directed mutagenesis

      • Identify nucleotide sequences in genes of interest

      • Create complementary DNA sequences in the lab

      • Insert sequences into target cells

      • Target cells swap sequences for introduced complementary sequences, creating recombinant DNA

    • Protoplast fusion

      • Scientists use enzymes to remove cell walls from multiple cells, creating protoplasts

      • The technique is widely used in fungi

  • Applications for Medicine

    • Diabetes insulin

    • Interferons

      • Body’s antiviral molecules used to treat infection

    • Gene Therapy

      • Inserting therapeutic DNA into patient cells

      • Gene silencing

      • Gene editing

        • CRISPR

    • Vaccines

  • Applications for Agriculture

    • Tumor Plasmid induces tumor formation in plant species

      • Cloning vectors that transfer genes from prokaryotes to eukaryotes

    • Biopesticides

  • Genomics

    • the study of gene organization, the information they store, and the gene products they code for

  • Genome

    • total DNA content of cells

  • Structural genomics

    • Study of physical nature of genomes

    • Human Genome Project

    • Bioinformatics

      • Genome study using computers

      • combines computer science, biology, statistics, and math

      • Annotation

        • technique used to determine the location of genes on a genome

  • Functional Genomics

    • Study of genome function-how genomes work

    • Coding sequences

      • Open reading frames that encode proteins

        • Paralogs

          • Two genes are paralogs if they consist of similar sequences and are found in the same genome

          • Arise due to gene duplication events

        • Orthologs

          • Consist of similar sequences are found in different organisms

            • In Silico Analysis

              • Computer analysis that allows researchers to further examine bioinformatics

        • Motifs

          • short amino acid patterns known to have specific functions

  • DNA Microarray

    • Observe DNA expression for thousands of genes simultaneously

  • Gel electrophoresis

    • Proteomics can be used to obtain information that mRNA cannot give us.

    • Involved in gel electrophoresis

  • Functional Proteomics

    • Provides information about what is actually going on in the cell

  • Structural Proteomics

    • The study of the three-dimensional structure of proteins

  • Two-Dimensional Gel Electrophoresis

    • Separates proteins based on charge and molecular mass

  • Metabolomics

    • Allows an assessment of a cell’s physiological status at any given point in time

  • Lipidomics

    • Study of the entire lipid profile, assess environmental impact on cell membranes

  • Comparative Genomics

    • Study of differences among genomes of different organisms

    • Has led to creation of new therapy targets and vaccine

    • Developed a therapy for leprosy

  • Metagenomics

    • Study of genetic material recovered from environmental samples

    • Bioprospecting

      • Search for species that have medical and commercial uses for humans

      • High-throughput sequencing technology

        • Allows researchers to rapidly and cheaply sequence and analyze large amounts of DNA