Chapter 10 Genetic Engineering A Revolution in Molecular

Chapter 10: Genetic Engineering. A Revolution in Molecular Biology

Basic Elements and Applications of Genetic Engineering • Basic science: when no product or application is directly derived from it • Applied science: useful products and applications that owe their invention to the basic research that preceded them • Six applications and topics in genetic engineering – – – Tools and techniques Methods in recombinant DNA technology Biochemical products of recombinant DNA technology Genetically modified organisms Genetic treatments Genome analysis

Tools and Techniques of Genetic Engineering • DNA: The Raw Material – Heat-denatured DNA • DNA strands separate if heated to just below boiling • Exposes nucleotides • Can be slowly cooled and strands will renature

Restriction Endonucleases • Enzymes that can clip strands of DNA crosswise at selected positions • Hundreds have been discovered in bacteria • Each has a known sequence of 4 to 10 pairs as its target • Can recognize and clip at palindromes

Figure 10. 1

• Can be used to cut DNA in to smaller pieces for further study or to remove and insert sequences • Can make a blunt cut or a “sticky end” • The pieces of DNA produced are called restriction fragments • Differences in the cutting pattern of specific restriction endonucleases give rise to restriction fragments of differing lengthsrestriction fragment length polymorphisms (RFLPs)

Ligase and Reverse Transcriptase • Ligase: Enzyme necessary to seal sticky ends together • Reverse transcriptase: enzyme that is used when converting RNA into DNA

Figure 10. 2

Analysis of DNA • Gel electrophoresis: produces a readable pattern of DNA fragments Figure 10. 3

Nucleic Acid Hybridization and Probes • Two different nucleic acids can hybridize by uniting at their complementary regions • Gene probes: specially formulated oligonucleotide tracers – Short stretch of DNA of a known sequence – Will base-pair with a stretch of DNA with a complementary sequence if one exists in the test sample • Can detect specific nucleotide sequences in unknown samples • Probes carry reporter molecules (such as radioactive or luminescent labels) so they can be visualized • Southern blot- a type of hybridization technique

Figure 10. 4

Probes Used for Diagnosis Figure 10. 5

Fluorescent in situ Hybridizaton (FISH) • Probes applied to intact cells • Observed microscopically for the presence and location of specific genetic marker sequences • Effective way to locate genes on chromosomes

Methods Used to Size, Synthesize, and Sequence DNA • Relative sizes of nucleic acids usually denoted by the number of base pairs (bp) they contain • DNA Sequencing: Determining the Exact Genetic Code – Most detailed information comes from the actual order and types of bases- DNA sequencing – Most common technique: Sanger DNA sequence technique

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Polymerase Chain Reaction: A Molecular Xerox Machine for DNA • Some techniques to analyze DNA and RNA are limited by the small amounts of test nucleic acid available • Polymerase chain reaction (PCR) rapidly increases the amount of DNA in a sample • So sensitive- could detect cancer from a single cell • Can replicate a target DNA from a few copies to billions in a few hours

Figure 10. 7

Three Basic Steps that Cycle • Denaturation – Heat to 94°C to separate in to two strands – Cool to between 50°C and 65°C • Priming – Primers added in a concentration that favors binding to the complementary strand of test DNA – Prepares the two strands (amplicons) for synthesis • Extension – 72°C – DNA polymerase and nucleotides are added – Polymerases extend the molecule • The amplified DNA can then be analyzed

Methods in Recombinant DNA Technology • Primary intent of recombinant DNA technology- deliberately remove genetic material from one organism and combine it with that of a different organism • Form genetic clones – Gene is selected – Excise gene – Isolate gene – Insert gene into a vector – Vector inserts DNA into a cloning host

Figure 10. 8