Chapter 15: DNA Technology and Genomics

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DNA Cloning

• Recombinant DNA technology isolates and amplifies specific sequences of DNA by incorporating them into vector DNA molecules.
  • Researchers then clone—propagate and amplify— the resulting recombinant DNA in organisms such as E. coli.
• Researchers use restriction enzymes to cut DNA into specific fragments.
  • Each type of restriction enzyme recognizes and cuts DNA at a highly specific base sequence.
  • Many restriction enzymes cleave DNA sequences to produce complementary, single-stranded sticky ends.
• Geneticists often construct recombinant DNA molecules by allowing the ends of a DNA fragment and a vector, both cut with the same restriction enzyme, to associate by complementary base pairing.
  • Then DNA ligase covalently links the DNA strands to form a stable recombinant molecule.
• A genomic DNA library contains cloned copies of thousands of DNA fragments that represent the total DNA of an organism.
• A cDNA library contains cloned copies of mature mrNA isolated from eukaryotic cells produced by using reverse transcriptase.
  • These copies, known as complementary DNA (cDNA), are incorporated into recombinant DNA vectors.
• Genes present in genomic DNA from eukaryotes contain introns, regions that do not code for protein.
  • Those genes can be amplified in bacteria, but the protein is not properly expressed.
  • Because introns have been removed from mature mrNA molecules, eukaryotic genes in cDNA libraries can be expressed in bacteria to produce functional protein products.
• Researchers use a radioactive DNA sequence as a DNA probe to screen thousands of recombinant DNA molecules in bacterial cells to find the colony that contains the DNA of interest.
• The polymerase chain reaction (PCR) is a widely used, automated, in vitro technique in which researchers use specific primers to target a particular DNA sequence.
  • The sequence is amplified by repeatedly heating the target DNA to separate the strands and then copying the single-stranded target sequences using a heat-resistant DNA polymerase.
• Using pCr, scientists amplify and analyze tiny DNA samples taken from various sites, from crime scenes to archaeological remains.

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Tools for Studying DNA

• A Southern blot detects DNA fragments by separating them using gel electrophoresis and then transferring them to a nitro-cellulose or nylon membrane.
  • A probe is then hybridized by complementary base pairing to the DNA bound to the membrane, and the band or bands of DNA are identified by autoradiography or chemical luminescence.
• When RNA molecules that are separated by electrophoresis are transferred to a membrane, the result is a Northern blot.
• A Western blot consists of proteins or polypeptides previously separated by gel electrophoresis.
• Bioinformatics software can be used to locate and compare the nucleotide sequence of newly cloned DNA sequences with related genes from other organisms.
  • Knowing the function of the gene in a related organism will provide insights into the function of the new gene.
• DNA and protein sequences in databases can be analyzed using bioinformatics software to identify and compare the structures and functions of proteins expressed in related organisms.
  • Northern blotting identifies a specific mRNA species in gel electrophoresis blots of mRNA isolated from different tissues using a radioactive probe.
• RT–PCR uses automated machines to measure PCR-amplified levels of cDNAs from reverse-transcribed mRNAs.
  • Many different mRNAs from different tissues can be measured simultaneously by using fluorescent primers specific for each target mRNA.
• DNA chips can compare mRNA levels over entire genomes.
  • The mRNAs from normal and diseased tissues are reverse transcribed to create different fluorescent color-coded cDNAs from each population.
  • The cDNAs are then annealed cDNAs in microdots that were synthesized using previously sequenced genomic DNA data.

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Genomics

• Studies such as the ENCODE project have shown that large regions of the genome that are devoid of protein-coding genes contain genes for non-protein-coding RNAs that are involved in the regulation of protein-coding genes.
  • Defects in these regions are associated with genetic markers for many types of genetic and cancer disease states.
• These methods prevent the expression and function of a targeted gene, allowing the investigator to observe its effects on the organism.
  • The connection of the silenced or knocked- out gene to a phenotypic defect indicates that the gene plays some essential role in that biochemical or developmental process.

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Applications of DNA Technologies

• Genetically altered bacteria produce many important human protein products, including insulin, growth hormone, tissue plasminogen activator (TPA), tissue growth factor-b (TGF-b), and clotting factor VIII.
• DNA fingerprinting is the analysis of an individual’s DNA.
  • It is based on a variety of short tandem repeats (STRs), molecular markers that are highly polymorphic within the human population.
  • DNA fingerprinting has applications in areas such as law enforcement, issues of disputed parentage, and tracking tainted foods.
• Transgenic organisms have foreign DNA incorporated into their genetic material.
  • Transgenic livestock produce foreign proteins in their milk.
  • Transgenic plants have great potential in agriculture.

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DNA Technology Has Raised Safety Concerns

• Some people are concerned about the safety of transgenic organisms.
  • To address these concerns, scientists carry out recombinant DNA technology under specific safety guidelines.
• The introduction of transgenic plants and animals into the natural environment, where they may spread in an uncontrolled manner, is an ongoing concern.

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