Exam 2: Ch 8 & 15: Genetic Engineering & Diagnosing Infections Flashcards
Genetic recombination
When DNA is transferred from one to another
Increases diversity in gene pool
End result is new strain different from both the donor and the original recipients
During meiosis of human gametes
Genetic recombination in bacteria
Bacteria only has asexual reproduction
They share DNA and pass genes back and forth which greatly increases the gene pool
Depends on the fact that bacteria have plasmids and are adept at interchanging genes
Provide genes for resistance to drugs and metabolic poisons, new nutritional and metabolic capabilities, and increased virulence and adaptation to the environment
Vertical gene transfer
from one organism to its offspring
Horizontal gene transfer
transferred btwn organisms
Antibiotic resistant (R) plasmids
some carry many antibiotic resistance genes; when bacteria collect many plasmids and these plasmids have many of those genes = “superbug”
3 types of genetic transfer
conjugation
transformation
transduction
Transposons
small segments of DNA that can be (be transposed) from one region of a DNA molecule to another (jumping genes)
Phenotypic methods for diagnosing microbial diseases
direct examination/observation of specimen
isolation media and morphological testing
Genotypic methods for diagnosing microbial diseases
genetic techniques increasingly being used as a sole resource for identifying bacteria
PCR
genetic probes
nucleic acid sequencing
fluorescent in situ hybridization
Immunological methods for diagnosing microbial diseases
serological testing
Direct examination/observation of specimen (phenotypic method)
stains (Gram and acid fast)
Isolation media and morphological testing (phenotypic methods)
Selective media – encourage growth only of the suspected pathogen
Differential media – used to identify definitive characteristics and fermentation patterns
Physiological/biochemical characteristics (phenotypic methods)
Enzyme production and assessing nutritional/metabolic activities (fermentation of sugars, capacity to digest polymers, production of gas, sensitivity to antibiotics, nutrient sources) Biochemical testing – enzyme mediated metabolic reactions visualized by a color change
Phage typing (phenotypic methods)
phage = always dealing w/viruses
Testing for sensitivity to various phage groups
Lawn of bacterial cells inoculated onto agar, mapped off into blocks, phage are exposed to each block
Clear areas corresponding to lysed cells = sensitivity to that phage
PCR (genotypic method)
bacterial DNA from a sample can be PCRed at a particular area in the genome; using restriction endonucleases the PCR produced are cut and an RFLP emerges – this is a DNA fragment of that bacteria
Genetic probes (genotypic method)
Hybridization:
Used to identify bacterial species by analyzing the sequences of nitrogenous bases in DNA
Probes: small fragments of single-stranded DNA or RNA complementary to the specific DNA sequence of a particular microbe
Probes with florescent or radioactive tags are added to the RFLP to visible changes in the DNA sequence in that area
Nucleic acid sequencing (genotypic method)
Best way to identify a bacterial species is to determine the 16s rRNA sequence of that bacteria
16s rRNA is part of the 30s subunit of the bacterial ribosome
16s rRNA is highly conserved across species and evolutionary time
Perfectly suited for bacterial identification and diagnosis of infection
Fluorescent in situ hybridization (genotypic method)
Fluorescent in situ hybridization (FISH) rapidly identifies 16s RNA sequences without first culturing the organism
Relies on dyes to emit visible light in response to UV radiation
Turnaround time for identifying suspect pathogens present in blood cultures has been reduced from 24 hours to 90 minutes
Electrophoresis
DNA separated according to size by running it thru a gel (made of agarose); the gel is placed in an electrical field that pushes the negatively charged DNA towards the positive electrode (DNA is negatively charged!!!!)
Restriction endonucleases
enzymes capable of recognizing foreign DNA and breaking the bonds btwn adjacent nucleotides on both strands of DNA
− Protects bacteria against incompatible DNA of bacteriophages
− Allows biotechnologists to cleave DNA at desired sites
− Necessary for recombinant DNA technology
− Recognize & clip at palindromes
− Cut 4-5 bases on the 3’ and 5’ strand, leaving overhangs on each end; adhesive tails will base-pair w/complimentary tails on other DNA fragments or plasmids
Restriction fragment length polymorphisms (RFLPs)
differences in the cutting patterns of specific restriction endonucleases; allows comparison of different cutting sites at specific areas in the genome
PCR
specific DNA replication of a particular portion of the DNA
3 steps
Recombinant DNA technology
Remove genetic material from one organism and combine it with that of a different organism
Bacteria can be genetically engineered to mass produce substances such as hormones, enzymes, and vaccines difficult to synthesize by usual industrial methods
Genetic clones and cloning
Genetic clones and cloning
Involves removal of a selected gene from an animal, plant, or microorganism and grow it in a host microorganism
Gene must be inserted into a vector (usually a plasmid or a virus)
Vector: inserts the gene into the cloning host
Cloning host: usually a bacterium or yeast which can translate the gene into the desired protein
Cloning vectors
-Small, well characterized, easy to manipulate
-Readily accepted DNA by the cloning host
-Contain an origin of replication; contain a selective antibiotic resistant gene
-Ex. Plasmids, phages
Producing recombinant DNA
Start with a cloning vector (special plasmid) and DNA with your gene of choice.
Cut the cloning vector and your desired gene out of the parent chromosome with specific enzymes.
Mix the vector and the gene together with a ligase enzyme which “seals” the DNA together.
Use various techniques to insert the vector +gene into a new cell.
Grow cell on selective or differential media to find out which cells possess the recombinant plasmid.
