Chapter 10 Genetic Engineering. A Revolution in Molecular Biology

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1 Chapter 10 Genetic Engineering A Revolution in Molecular Biology

2 Genetic Engineering Bioengineering The direct and deliberate modification of an organism s genome Biotechnology The use of an organism s biochemical and metabolic pathways for industrial production of materials

3 Tools and techniques of Genetic Engineering Practical Properties of DNA Intrinsic properties of DNA hold true even in a test tube DNA heated from 90 C to 95 C; the two strands separate. The nucleotides can be identified, replicated, or transcribed. Heating Cooling Slowly cooling the DNA allows complementary nucleotides to hydrogen bond and the DNA will regain double-stranded form.

4 Enzymes for Dicing, Splicing, and Reversing Nucleic Acids Restriction endonucleases recognize specific sequences of DNA and break phosphodiester bonds between adjacent nucleotides The enzymes can be used to cleave DNA at desired sites Recognize and clip the DNA at palindrome base sequences Restriction fragments: Smaller pieces of DNA formed by endonuclease action Endonuclease EcoRI HindIII HaeIII Cutting pattern G A A T T C A A G C T T G G C C C T T A A G T T C G A A C C G G

5 Restriction endonuclease digestion Restriction endonucleases cleave DNA at specific recognition sites, which are usually 4 to 6 bp and palindromes. May generate blunt ends or staggered ends.

6 Restriction endonuclease digestion Agarose gel electrophoresis can be used to analyze the DNA fragments obtained by treatment with restriction enzymes. Heavier fragments run slower DNA is negatively charged

7 Restriction Fragment Length Polymorphisms (RFLPs) DNA sequences vary, even among members of the same species Differences in the cutting pattern of specific restriction endonucleases give rise to fragments of differing lengths (RFLPs)

8 Enzymes for Dicing, Splicing, and Reversing Nucleic Acids Ligase : rejoins phosphate-sugar bonds (sticky ends) cut by restriction endonucleases Used for final splicing of genes into plasmids and chromosomes DNA from organism 1 DNA from organism 2

9 Enzymes for Dicing, Splicing, and Reversing Nucleic Acids Reverse transcriptase : makes a DNA copy of RNA cdna cdna can be made from mrna, trna, or rrna Provides a means of synthesizing eukaryotic genes from mrna transcripts synthesized gene is free of introns

10 Synthesis of cdna from eukaryotic mrna

11 Methods for DNA analysis Gel electrophoresis - separates DNA fragments based on size DNA samples are placed on soft agar gel and subjected to an electric current DNA for sample 3 Restriction endonucleases selectively cleave sites of DNA ( ) Wells Gel (+) DNA migrates toward positive electrode

12 Methods for DNA analysis Negative charge of DNA causes it to move toward positive pole Rate of movement is dependent on size of fragment larger fragments move more slowly DNA migrates toward positive electrode

13 Methods for DNA analysis Fragments are stained with Ethidium Bromide for observation Useful in characterizing DNA fragments and comparing for genetic similarities Well Samples Water Known DNA size markers

14 Methods for DNA analysis Nucleic acid hybridization and probes Single-stranded DNA can unite with other single-stranded DNA or RNA, and RNA can unite with other RNA HYBRIDIZATION Hybridization: DNA: DNA, DNA: RNA, RNA: RNA Hybridization is the foundation for gene probes Gene probes: short DNA fragments of a known sequence that will base-pair with a stretch of DNA with a complementary sequence ACGTCGCGCTTGGCAC CAGCGCGA Useful in detecting specific nucleotide sequences in unknown samples

15 Southern blot method 1) DNA is digested with a restriction enzyme 2) separated by gel electrophoresis on an agarose gel Because there are so many different restriction fragments on the gel, it usually appears as a smear rather than discrete bands. 3) DNA is denatured into single strands by incubation with NaOH 4) DNA is transferred to a membrane which is a sheet of special blotting paper. The DNA fragments retain the same pattern of separation they had on the gel. This is referred to as a blot. 5) Blot is incubated with radioactive probe which is single-stranded DNA. Probe will form base pairs with its complementary DNA sequence form a double-stranded DNA molecule. 6) The location of the probe is revealed by radiation that will darken a X-ray film

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17 Methods for DNA analysis Fluorescent in situ hybridization (FISH) - probes are applied to intact cells and observed for the presence and location of specific sequences Useful for diagnostics or locating genes on a chromosome

18 Methods used to size, synthesize, and sequence DNA DNA sequencing determining the actual order and type of bases for all types of DNA Most common sequencing technique is Sanger technique Test strands are denatured to serve as a template to synthesize complementary strands Fragments are divided into tubes that contain primers, DNA polymerase, all 4 nucleotides, and fluorescent labeled dideoxynucleotide

19 Sanger sequencing method

20 Sanger sequencing Rajesh Ramakrishnan, PhD dissertation, University of Arizona

21 The Omics era Genomics - the systematic study of an organism s genes and their functions Proteomics - the study of an organism s complement of proteins and functions mediated by the proteins Metagenomics - the study of all the genomes in a particular ecological niche, as opposed to individual genomes from single species Metabolomics - the study of the complete complement of small chemicals present in a cell at any given time

22 Methods used to size, synthesize, and sequence DNA Polymerase Chain Reaction (PCR) method to amplify DNA; rapidly increases the amount of DNA in a sample Primers of known sequence are added, to indicate where amplification will begin, along with special heat tolerant DNA polymerase and nucleotides Repetitively cycled through denaturation, priming, and extension Each subsequent cycle doubles the number of copies for analysis Essentially important in gene mapping, the study of genetic defects and cancer, forensics, taxonomy, and evolutionary studies

23 Polymerase Chain Reaction (PCR) The amplification rate is exponential How many copies of DNA will be present after 5 cycles of PCR if: 1) you started with one DNA molecule then, 1 x 2^5 = 32 2) you started with two DNA molecules then, 2 x 2 ^5 = 64

24 Methods in recombinant DNA technology Recombinant DNA technology the intentional removal of genetic material from one organism and combining it with that of a different organism Objective of recombinant technology is cloning which requires that the desired donor gene be selected, excised by restriction endonucleases, and isolated The gene is inserted into a vector (plasmid, virus) that will insert the DNA into a cloning host Cloning host is usually bacterium or yeast that can replicate the gene and translate it into a protein product

25 Recombinant DNA technique

26 Characteristics of cloning vectors Must be capable of carrying a significant piece of donor DNA Must be readily accepted by the cloning host Plasmids small, well characterized, easy to manipulate and can be transferred into appropriate host cells through transformation Bacteriophages have the natural ability to inject their DNA into bacterial hosts through transduction

27 Vector considerations Origin of replication is needed so it will be replicated Vector must accept DNA of the desired size Gene which confers drug resistance to their cloning host

28 Desirable features in a cloning host

29 1) A plasmid vector is cut with a restriction endonuclease (EcoRI) that produces sticky ends. Construction of recombinant DNA 2) The same enzyme is used to digest foreign DNA, producing a series of fragments, all with identical sticky ends. 3) The cut vector and foreign DNA fragments are mixed. 4) Cohesive ends of the foreign DNA and vector anneal to form a chimeric molecule containing two nicks. 5) DNA ligase is used to seal the nicks and connect the phosphodiester backbones.

30 Protein production by genetic engineering The recombinant is introduced into a cloning host The cloning host transcribes the gene and translates the protein

31 Screening for recombinant microbes Use selective media to quickly identify recombinants Bacteria lack plasmid Culture of cloning host after incubation with recombinant plasmid Bacteria with recombinant plasmid (1) (2) Bacteria carrying plasmid Ampicillinresistance gene Regular nonselective medium with two types of colonies Selective medium with ampicillin Pure culture of bacteria containing cloned gene

32 Blue white screening for recombinant microbes Bacteria transformed with original pblu: Blue colonies on amp + x-gal Bacteria transformed with pblu + insert DNA: White colonies on amp + x-gal Rajesh Ramakrishnan, PhD dissertation, University of Arizona

33 Biochemical products of recombinant DNA technology Enables large scale manufacturing of life-saving hormones, enzymes, vaccines Insulin for diabetes Erythropoietin for anemia Factor VIII for hemophilia HBV vaccine

34 Examples of recombinant technology products

35 Genetically Modified Organisms (GMO, transgenic) Recombinant microbes Bacillus thuringienisis encodes an insecticide Many enzymes, hormones, and antibodies used in drug therapy are manufactured using mammalian cell culture Cell cultures can modify the proteins Microbes to bioremediate polluted environments Agrobacterium tumefaciens: a natural plant tumor-producing bacterium - Ti plasmid inserts into the genomes of the infected plant cells

36 Transgenic animals Use a virus to transfect a fertilized egg or early embryo Transgenic animals will transcribe and translate eukaryotic genes Transgenic animals will transcribe and translate eukaryotic genes

37 Transgenic mice expressing Green fluorescent protein Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett May 5;407(3):313-9

38 Examples of transgenic animals

39 Examples of transgenic plants

40 Genetic Treatments: Introducing DNA into the body Gene therapy: correct or repair a faulty gene in humans Ex vivo therapy: normal gene cloned in vectors, tissue removed from the patient In vivo therapy: naked DNA or vector is directly introduced into the patient s tissues 1 (1) 1 Normal gene is isolated from healthy subject. 2 Gene is cloned Gene is inserted into retrovirus vecto Bone marrow sample is taken from patient with genetic defect. Stem cells from marrow are infected with retrovirus, (enlarged view) Transfected cells (red dots) are infused into patient. Patient is observed for expression of normal gene Marrow stem cell 4 3

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43 DNA technology as genetic medicine Gene silencing techniques by RNA interference sirna: has bases complementary to the sense strand of mrna Degrades mrna or prevents mrna translation Ramakrishnan R, Liu H, Donahue H, Malovannaya A, Qin J, Rice AP. Identification of novel CDK9 and Cyclin T1-associated protein complexes (CCAPs) whose sirna depletion enhances HIV-1 Tat function. Retrovirology Oct 30;9:90.

44 Genome analysis DNA Fingerprinting Every individual has a unique sequence of DNA Methods used include 1) restriction endonucleases, 2) electrophoresis, 3) hybridization, and 4) Southern blot

45 Genome analysis DNA Fingerprinting is used to Identify hereditary relationships Study inheritance of patterns of diseases Study human evolution Identify criminals or victims of disaster Analysis of mitochondrial DNA is used to trace evolutionary origins

46 Genome Analysis Microarray analysis track the expression of thousands of genes used to identify and devise treatments for diseases based on the genetic profile of the disease Ramakrishnan R, Donahue H, Garcia D, Tan J, Shimizu N, Rice AP, Ling PD. Epstein-Barr virus BART9 mirna modulates LMP1 levels and affects growth rate of nasal NK T cell lymphomas. PLoS One. 2011;6(11):e27271.

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