MID 2 - Lecture 8

Question 1

Antibiotic resistance: 2 mechanisms

Answer 1

1. spontaneous mutation
2. Horizontal gene transfer

Question 2

Spontaneous mutations

Answer 2

arise without exposure to external agents, point mutations (most common - small), large not common, results from errors in DNA rep

Question 3

Horizontal gene transfer

Answer 3

transfer of genes from one independent mature organism to another, DNA can replicate or integrate into genome

Question 4

HGT steps

Answer 4

-Uptake of DNA from environment
a. uptake of naked DNA by cell
b. uptake of DNA plasmid
-conjugation : transfer of DNA between cells by direct contact (sex pilius)
-transduction : transfer of bacterial genes by viruses, lytic cycle integrate into host genome

Question 5

F factor

Answer 5

100 000 bp long plasmid, genes responsible for cell attachment and plasmid transfer, cell containing f factor is F+, without is F-

Question 6

Sex pilus

Answer 6

attach f+ to f- for DNA transfer (conjugation), DNA is single stranded

Question 7

Hfr conjugation

Answer 7

F factor in chromosome rather than plasmid form
- f factor attempts to replicate, copies surrounding DNA and transfers to recipient
- recombination of chromosomal genes into recioient occurs 1000x more often with Hfr
- recombination leads to variation

Question 8

F’ conjugation (excises from host)

Answer 8

f factor excised from host AND brings host genes, f factor plasmid now called f’, host genes now transferred to second host by conjugation

Question 9

R plasmids (resistance)

Answer 9

carry genes required for resistance to antibiotics, discovered in shigella strains, replicate autonomously and have genes for transfer by conjugation, transferred between species (related and unrelated)

Question 10

Generalized transduction (phage mistake)

Answer 10

during lytic cycle, during viral assembly fragments of host DNA mistakenly packaged into phage head, DNA can be transferred to new host cell and incorporated into genome

Question 11

Specialized transduction (prophage)

Answer 11

carried out only by temperate phages (have established lysogeny), prophage is incorrectly excised, small portion of bacterial genome is transferred along with phage genome, can contain resistance gene

Question 12

Genomics

Answer 12

study of genomes, their molecular content, and gene products they encode

Question 13

Metagenomics (definition)

Answer 13

study of all organisms present into an environmental samples without culturing (gut, soil)

Question 14

DNA (RNA) sequencing platforms

Answer 14

sanger (medium reads), Next generation: illumina (short), ion torrent (medium), third generation: pacbio (long), nanopore (very long)

Question 15

Sanger DNA sequencing

Answer 15

First, also chain termination method, uses ddNTP, further dNTPS cannot be added, blocks chain elongation

Question 16

Original sanger method

Answer 16

mix strands of DNA: template, primer, DNA polymerase I, 4 deoxynucleotides, small amount of one ddNTP, DNA synthesis occurs - random insertion of ddNTP generates DNA fragment of diff lengths

Question 17

Sanger (old way)

Answer 17

4 rxns (diff ddNTP), fragments in each reaction mixture separated on DNA gels, gel autoradiographed and sequence read manually based on size of fragments

Question 18

Sanger (current) - laser beams

Answer 18

4 colour dyes (no ddNTP), mixed in same tube, electrophoresis and laser beams determine order

Question 19

Metagenomics

Answer 19

cultivation independent (no media), diversity and metabolic potential of microbial commuities, NGS

Question 20

NGS (short)

Answer 20

faster, cheaper, decreases likelihood of false sequence data (no random mistakes), short, sheared pieces of DNA to solid substrate, avoid need to insert DNA frag into vectors

Question 21

Reversible chain termination sequence

Answer 21

NGS method, fluroescent nucleotides (stop DNA copying camera takes picture), stop can be removed and put back, 75-150 bases

Question 22

Sequencing by synthesis

Answer 22

identifies each nucleotide as its incorporated, uses fluroscent nucleotide that stops rxn, nucleotide does not fluoresce until incorporated into DNA strand

Question 23

Ion torrent (short)

Answer 23

sequencing by synthesis, completementary strand built upon template, no modified nucleotides or optics, additon of dNTP releases H+ (pH change), 99.6 accuracy, expensive, fast

Question 24

pacbio- SMRT sequencing

Answer 24

single molecule, real time, cell contains million of zero-mode wave guides, single fixed DNA polymerase in ZMW extends template, bases are fluroscent and read by detector, 30 000 bp in length, 87% accurate

Question 25

Oxford nanopore (determines sequence of bases)

Answer 25

tiny holes in a chip, ionic current passed through, protein nanopore electrically resistant polymer membrane, DNA translocated through pore by enzyme motor, simple, small, connected to laptop, 2 000 000 bps read

Question 26

MinION, gridION, PromethION

Answer 26

mini: pocket sized, 512 nanopore channels
Grid: 5 minion flow cells
prometh: 48 flow cells of 3000 nanopore channels