Microbiology: A Systems Approach, 2nd ed.

Chapter 10: Genetic Engineering- A Revolution in Molecular Biology

10.1 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

10.2 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

10.3 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

Technical Aspects of Recombinant DNA and Gene Cloning

• Strategies for obtaining genes in an isolated state– DNA removed from cells, separated into

fragments, inserted into a vector, and cloned; then undergo Southern blotting and probed

– Gene can be synthesized from isolated mRNA transcripts

– Gene can be amplified using PCR• Once isolated, genes can be maintained in a

cloning host and vector (genomic library)

Characteristics of Cloning Vectors

• Capable of carrying a significant piece of the donor DNA

• Readily accepted by the cloning host• Must have a promoter in front of the cloned gene• Vectors (such as plasmids and bacteriophages)

should have three important attributes:– An origin of replication somewhere on the vector– Must accept DNA of the desired size– Contain a gene that confers drug resistance to their

cloning host

Figure 10.9

Characteristics of Cloning Hosts

Construction of a Recombinant, Insertion into a Cloning Host, and

Genetic Expression

Figure 10.10

Figure 10.11

Synthetic Biology: Engineering New Genetic Capabilities

• Scientists are attempting to create microbes that produce hydrogen as fuel

• Can use recombinant techniques mentioned previously

10.4 Biochemical Products of Recombinant DNA Technology

10.5 Genetically Modified Organisms

• Transgenic or genetically modified organisms (GMOs): recombinant organisms produced through the introduction of foreign genes

• These organisms can be patented

Recombinant Microbes: Modified Bacteria and Viruses

• Genetically altered strain of Pseudomonas syringae– Can prevent ice crystals from forming– Frostban to stop frost damage in crops

• Strain of Pseudomonas fluorescens– Engineered with a gene from Bacillus thuringiensis – Codes for an insecticide

• Drug therapy• Bioremediation

Transgenic Plants: Improving Crops and Foods

• Agrobacterium can transfect host cells• This idea can be used to engineer plants

Figure 10.12

Transgenic Animals: Engineering Embryos

• Several hundred strains have been introduced• Can express human genes in organs and organ

systems• Most effective way is to use viruses

Figure 10.13

10.6 Genetic Treatments: Introducing DNA into the Body

• Gene Therapy– For certain diseases, the phenotype is due to the

lack of a protein– Correct or repair a faulty gene permanently so it

can make the protein– Two strategies

• ex vivo• in vivo

Figure 10.14

in vivo

• Skips the intermediate step of incubating excised patient tissue

• Instead the naked DNA or a virus vector is directly introduced into the patient’s tissues

DNA Technology as Genetic Medicine

• Some diseases result from the inappropriate expression of a protein

• Prevent transcription or translation of a gene

Antisense DNA and RNA: Targeting Messenger RNA

• Antisense RNA: bases complementary to the sense strand of mRNA in the area surrounding the initiation site– When it binds to the mRNA, the dsRNA is inaccessible to the

ribosome– Translation cannot occur

• Single-stranded dNA usually used as the antisense agent (easier to manufacture)

• For some genes, once the antisense strand bound to the mRNA, the hybrid RNA was not able to leave the nucleus

• Antisense DNA: when delivered into the cytoplasm and nucleus, it binds to specific sites on any mRNAs that are the targets of therapy

Figure 10.15

10.7 Genome Analysis: Maps, Fingerprints, and Family Trees

• Possession of a particular sequence of DNA may indicate an increased risk of a genetic disease

• Genome Mapping and Screening: An Atlas of the Genome– Locus: the exact position of a particular gene on a chromosome– Alleles: sites that vary from one individual to another; the types

and numbers are important to genetic engineers– Mapping: the process of determining location of loci and other

qualities of genomic DNA• Linkage maps: show the relative proximity and order of genes on a

chromosome• Physical maps: more detailed arrays that also give the numerical size

of sections in base pairs• Sequence maps: produced by DNA sequencers

– Genomics and bioinformatics: managing mapping data

DNA Fingerprinting: A Unique Picture of a Genome

• DNA fingerprinting: tool of forensic science• Uses methods such as restriction

endonucleases, PCR, electrophoresis, hybridization probes, and Southern blot technique

Figure 10.16

Microarray Analysis

• Allows biologists to view the expression of genes in any given cell

Figure 10.17