Microbiology Chapter 10: Genetic Analysis and Genetic Engineering PDF

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Chapter 10: Genetic Analysis and Genetic E Engineering ngineering 10.1 Tools and Techniques of Genetic Engineering Basic science: no product or application is directly derived from it Genetic technology have assissted us in: ● Identifying criminals in courts ● Manufacturing human proteins through genetic engineering ● “Fixing” diseases using small RNA regualtory molecules

DNA: The Raw Material ● When DNA is heated at 90 to 95 °C, the two strands separate, exposing the info contained in their bases ● With nucleotides exposed, DNA can be easily identified, replicated, or transcribed ● As it cools, complementary nuclotides will hydrogen bond with each other and the strands will regain their familiar double-stranded form ● This is used in the polymerase chain reaction and use of nucleic acid probes

Systems for Dicing, Splicing, and Reversing Nucleic Acids The strands of DNA can be clipped crosswise at selected positions by means of enzymes called restriction endonucleases. ● Discovered in 1971 ● Found in bacterial and archaeal cells ● Recognize foreign DNA and break the bonds between certain nucleotides break the strand and protect the cell against bacteriophages or plasmids ● In lab, we use them to cleave DNA at specific sites Thousands of restriction endonucleases have been discovered in bacteria and archaea. ● Each type has a known sequence of 4 to 10 base pairs as its target ● Many have the property of recognizing and clipping at base sequences called palindromes ○ Palindromes are sequences of DNA that are identical when read from the 5’ to 3’ direction on both strands Endonucleases are named by combining the first letter of the bacterial genus, the first two letters of the species, and the endonuclease number. ● Ex: Eco RI is the first endonuclease found in E. coli Endonucleases are used in the lab to cut DNA into smaller pieces to study, and to remove/insert sequences during recombinant DNA techniques. ● Usually the enzymes make staggered symmetrical cuts that leave short tails called “sticky ends.” ○ The enzymes cut 4 or 5 bases on the 3’ and 5’ strand, leaving overhangs. This makes adhesive tails that will base-pair with complementary tails on

other DNA fragments. ○ Makes it possible to splice genes into specific sites ● These pieces are called restriction fragments. ○ Since DNA sequences can vary within the same species, there can be differences in length of these fragments, called restriction fragment length polymorphisms (RFLPs) ● Another enzyme, called a ligase, seals the sticky ends together by rejoining the phosphate-sugar bonds cut by endonucleases. ○ Its main application is in final splicing of genes into plasmids and chromosomes. An enzyme called reverse transcriptase is best known for its role in the replication of HIV and other retroviruses. ● It can help convert RNA into DNA ● Complementary DNA (cDNA) can be made from different forms of RNA ○ Allows us to synthesize a eukaryotic gene from mRNA ■ One advantage is that there will be no introns CRISPR (clustered regularly interspaced short palindromic repeats) is another system that alters genomes. ● In bacteria and archaea ● Includes an enzyme that is capable of recognizing and cutting foreign DNA (bacteriophages or plasmids), keeping the bacterium or archaea from being invaded. ○ They “learn” the identity of an attacking phage, by placing bits of its DNA in its own genome and can cut it up before it causes trouble ● Highly adapbtable for lab use. Cheap, easy to perform, and powerful.

Analysis of DNA Gel Electrophoresis One way to produce a readable pattern of DNA fragments is through gel electrophoresis. Samples are placed in compartments (wells) in a soft agar gel and subjected to an electrical current. ● The phosphate groups in DNA give the molecule a negative charge, which causes the DNA to move toward the positive pole in the gel. ● Rate of movement is based on the size of the fragments. ○ Longer fragments move more slowly and remain near the top of the gel.

Nucleic Acid Hybridization and Probes

Two different nucleic acids can hybridize by uniting at their complementary regions. All different combinations are possible: single stranded DNA w/ other single stranded DNA/RNA, or RNA w/ other RNA. ● Allowed for the development of formulated tracers called gene probes ○ A probe w/ a known sequence of DNA will base-pair w/ a complementary sequence if it exists in a test sample ■ Area of hybridization can be visualized as the probes have fluorescent dyes Probes are used for diagnosing the cause of an infection and for identifying a culture of a bacterium or virus. ● A method called a hybridization test does not require electrophoresis. ○ DNA is isolated, denatured, and placed on an absorbent filter, and then a solution containing a probe is added. ○ The blot is then developed and observed for areas of hybridization. With another method, fluorescent in situ hybridization (FISH), probes are applied to cells and observed for the presence and location of specific genetic sequences. ● Can be used to identify unknown bacteria living in their natural habitat

Polymerase Chain Reaction: A Molecular Xerox Machine for DNA The polymerase chain reaction (PCR) amplifies DNA and RNA for analysis. ● Increases the amount of DNA w/o the need for growing cultures or carrying out complex purification techniques. ● Developed in 1983 Initiation requires certain ingredients: ● Special DNA polymerases from thermophilic bacteria ○ Taq polymerase from Thermus aquaticus and Vent polymerase from Thermococcus litoralis ● A thermal cycler which will automatically perform the cyclic temperature changes There are three basic steps: ● Denaturation ● Priming ● Extension

● Usually carried out for 30-40 cycles ● Has a role in: ○ Gene mapping ○ Study of genetic defects and cancer ○ Forensics ○ Infectious disease diagnosis

Methods in Recombinant DNA Technology: How to Imitate Nature The primary intent of recombinant DNA technology is to purposely remove genetic material from one organism and combine with that of a different organism. ● Forms genetic clones. Cloning involves the removal of a selected gene from an animal, a plant, or microorganism followed by propagating it in a different host organism. ○ Remove a gene from a donor using restriction endonucleases ○ Insert the gene into a vector (usually plasmid or virus) ○ Vector will then insert it into a cloning host (usually bacterium or yeast) ○ The cloning host can replicate the gene and translate it into a protein The first steps are to locate its exact site on the donor and then to isolate it.

● DNA is removed from cells and separated into fragments by endonucleases. The right fragment is identified through a screening process, or ● A gene can be synthesized from mRNA transcripts using reverser transcriptase (cDNA), or ● A gene can be amplified using PCR Genomic libraries: collections of cDNA clones that represent the entire genome of numerous organisms.

Cloning Vectors Isolated genes are not manipulated on their own. They are spliced into a cloning vector, using restriction enzymes. ● Plasmids are good vectors because they are small, well characterized, easy to manipulate, and can be transferred into appropriate host cells through transformation ● Bacteriophages are also good because they have the ability to inject DNA into bacterial hosts through transduction All vectors have 3 attributes to consider: ● An origin of replication (ORI) is needed somewhere on the vector so it will be replicated by the DNA polymerase of the cloning host ● Must accept DNA of the desired size ● Usually have a gene that confers drug resistance to the cloning host

Cloning Hosts The best cloning hosts have several key characteristics. The traditional cloning host is E. coli.

10.2 Products of Recombinant DNA Technology Recombinant DNA technology can also be used to create sources of protein products or nucleotide sequences. It is used by pharmaceutical companies to manufacture medications.

Genetically Modified Organisms Recombinant organisms produced through the introduction of foreign genes are called transgenic or genetically modified organisms (GMOs). ● Recombinant Microbes: modified bacteria and viruses ○ Genetic alteration has created a different strain that prevents ice crystals from forming ○ Another strain has been engineered with a bacterium that codes fro an insecticide ● Synthetic Biology ○ Creating molecules and organisms from scratch

10.3 Genetic Treatments: Introducing DNA into the Body Gene Therapy Gene therapy: correct or repair a faulty gene in humans suffering from a fatal or debilitating disease ● Recombinant human insulin was the first genetically engineered drug to be approved for use in humans ● Various strategies: ○ Gene is cloned in vectors like retroviruses or adenoviruses ○ Patients’ tissues can be incubated with modified viruses to transfect them

w/ the gene

● Use of micro RNAs to either silence gene expression or inhibiting out-of-control miRNAs is currently being researched ● CRISPR techniques have begun to be developed to correct genetic defects

10.4 DNA Analysis: Maps and Profiles There are multiple ways to analyze DNA: ● DNA sequencing ● DNA profiling

Genome Sequencing Most detailed maps of a genome are sequence maps, which give an order of bases in a plasmid, a chromosome, or an entire genome. ● Showed how similar the genomes of unrelated organisms are ○ Humans share 80% withmice, 60% with rice, and 30% w/ the worm C. elegans Shotgun sequencing is an older method as follows:

Profiling ● Uses RFLPs to differentiate between organisms ● Individual traits could be due to SNPs (single nucleotide polymorphism) ○ Due to a point mutation ○ These could be used to customize medical treatment

Microarrays Microarrays are able to track the expression of thousands of genes at once ● Uses a “chip” (glass, silicon, or nylon) to which sequences of thousands of genes have been bound ● Fluorescently labeled cDNA is added to chip ● If the cDNA binds, it can be excited w/ a laser and then detected by computer...