Exam 2: Ch 8 & 15: Genetic Engineering & Diagnosing Infections

Question 1

Genetic recombination

Answer 1

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

Question 2

Genetic recombination in bacteria

Answer 2

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

Question 3

Vertical gene transfer

Answer 3

from one organism to its offspring

Question 4

Horizontal gene transfer

Answer 4

transferred btwn organisms

Question 5

Antibiotic resistant (R) plasmids

Answer 5

some carry many antibiotic resistance genes; when bacteria collect many plasmids and these plasmids have many of those genes = “superbug”

Question 6

3 types of genetic transfer

Answer 6

conjugation
transformation
transduction

Question 7

Transposons

Answer 7

small segments of DNA that can be (be transposed) from one region of a DNA molecule to another (jumping genes)

Question 8

Phenotypic methods for diagnosing microbial diseases

Answer 8

direct examination/observation of specimen

isolation media and morphological testing

Question 9

Genotypic methods for diagnosing microbial diseases

Answer 9

genetic techniques increasingly being used as a sole resource for identifying bacteria

PCR
genetic probes
nucleic acid sequencing
fluorescent in situ hybridization

Question 10

Immunological methods for diagnosing microbial diseases

Answer 10

serological testing

Question 11

Direct examination/observation of specimen (phenotypic method)

Answer 11

stains (Gram and acid fast)

Question 12

Isolation media and morphological testing (phenotypic methods)

Answer 12

Selective media – encourage growth only of the suspected pathogen
Differential media – used to identify definitive characteristics and fermentation patterns

Question 13

Physiological/biochemical characteristics (phenotypic methods)

Answer 13

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

Question 14

Phage typing (phenotypic methods)

Answer 14

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

Question 15

PCR (genotypic method)

Answer 15

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

Question 16

Genetic probes (genotypic method)

Answer 16

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

Question 17

Nucleic acid sequencing (genotypic method)

Answer 17

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

Question 18

Fluorescent in situ hybridization (genotypic method)

Answer 18

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

Question 19

Electrophoresis

Answer 19

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!!!!)

Question 20

Restriction endonucleases

Answer 20

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

Question 21

Restriction fragment length polymorphisms (RFLPs)

Answer 21

differences in the cutting patterns of specific restriction endonucleases; allows comparison of different cutting sites at specific areas in the genome

Question 22

PCR

Answer 22

specific DNA replication of a particular portion of the DNA

3 steps

Question 23

Recombinant DNA technology

Answer 23

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

Question 24

Genetic clones and cloning

Answer 24

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

Question 25

Producing recombinant DNA

Answer 25

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.