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 lengths- restriction 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

Figure 10.6

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