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General Microbiology > Ch.13 Modern Applications of Microbial Genetics > Flashcards

Ch.13 Modern Applications of Microbial Genetics Flashcards

1
Q

Molecular Cloning using Transformation Protocol

A

1) bacterial cells plated onto antibiotic-containing medium (inhibits cells that were not transferred by plasmid)
2) blue-white screening technique is used for lac Z encoding plasmid vectors (pUC19)
3) White colonies resulting from blue-white screening contain plasmids with an insert and can be further screened to characterize the foreign DNA
4) to ensure the correct DNA was incorporated into the plasmid, the DNA insert can then be sequenced

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2
Q

Blue colonies

A

have a functional beta-galactosidase enzyme because the lacZ gene is uninterrupted (no DNA inserted to polylinker site)
-result from digested, linearized plasmid religating to itself

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3
Q

White colonies

A

lack a functional beta-galactosidase enzyme indicating the insertion of foreign DNA within the polylinker site of the plasmid vector
- disrupts the lacZ gene

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4
Q

transfection

A

the introduction of recombinant DNA molecules into eukaryotic hosts

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5
Q

What laid the foundation for recombinant DNA technology?

A

use of bacterial hosts – particularly eukaryotic

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6
Q

what are transgenic plants & provide example

A

genetically engineered plants
- bacterial strain that produces human insulin

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7
Q

Flavr Savr plant

A

first transgenic plant sold commercially
- delayed ripening tomato in 1944

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8
Q

blue-white screening

A

allows selection of bacterial transformants that contain recombinant plasmids using the phenotype of a reporter gene that is disabled by insertion of the DNA fragment

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9
Q

biotechnology

A

the science of using living systems to benefit humankind

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10
Q

genetic engineering

A

involves the use of recombinant DNA technology, the process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material – the recombinant DNA is then introduced into a host organism

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10
Q

genetic engineering

A

involves the use of recombinant DNA technology, the process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material – the recombinant DNA is then introduced into a host organism

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11
Q

When is an organism considered transgenic?

A

if the DNA that is introduced comes from a different species

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12
Q

How do prokaryotes acquire foreign DNA and incorporate functional genes into their own genome?

A

Horizontal Gene Transfer processes…
- Conjugation: mating with other cells
- Transduction: viral infection
- Transformation: taking up DNA from environment

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13
Q

molecular cloning

A

a set of methods used to construct recombinant DNA and incorporate it into a host organism
-Herbert Boyer and Stanley Cohen first demonstrated cloning process with African clawed frog

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14
Q

How are DNA molecules manipulated in recombinant DNA technology?

A

using naturally occurring enzymes derived from bacteria and viruses

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15
Q

What makes the creation of recombinant DNA molecules possible?

A

endonucleases (restriction enzymes)
- bacterial enzymes produces as a protection mechanism to cut and destroy foreign DNA that is most commonly a result of bacteriophage infection

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16
Q

restriction enzyme role in forming recombinant molecules

A

cutting DNA fragments that can then be spliced into another DNA molecule
-cuts at a recognition site (usually palindromic sequence)

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17
Q

Palindrome

A

a sequence of letter that reads the same forward as backward
-contain same base pair sequences in 5-3 direction as the 5-3 direction on complementary sequence

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18
Q

How does a restriction enzyme cut the Palindrome?

A

it recognizes the palindrome and cuts each backbone at identical positions
-their cut reveals sticky or blunt ends

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19
Q

Do blunt ends or sticky ends of the palindrome better attach?

A

sticky ends: form hydrogen bonds b/w complementary bases

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20
Q

hybridization

A

refers to the joining together of two complementary single strands of DNA

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21
Q

What process rejoins the two single strands of DNA together in recombination?

A

ligation by DNA ligase

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22
Q

Plasmid

A

small pieces of circular, double stranded DNA that replicate independently of the bacterial chromosome

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23
Q

Vectors & Plasmid role

A

DNA molecules that carry DNA fragments from one organism to another
-ex) Plasmid vectors can be modified to contain genes to confer antibiotic resistance

24
Q

Polylinker site

A

a multiple cloning site (MCS); a short sequence containing multiple unique restriction enzyme recognition sites used for inserting DNA into the plasmid after restriction
-plasmid vectors used for cloning generally have a poly linker site

25
Q

What makes a plasmid vector versatile?

A

having multiple restriction enzyme recognition sites within the poly linker site
- allows for many different cloning experiments involving different restriction enzymes

26
Q

Where is the poly linker site found?

A

within a reporter gene

27
Q

Reporter gene & most common example in plasmid vectors

A

a gene sequence artificially engineered into the plasmid that encodes a protein that allows for visualization of DNA insertion
-can show whether the host cell contains the non-recombinant or recombinant vector
-MOST common: lacZ gene

28
Q

Bacterial LacZ gene

A

encodes beta-galactosidase enzyme which naturally degrades lactose

29
Q

Microarray Analysis

A

capitalizes on the hybridization between complementary nucleic acid sequences
-useful for comparison of gene-expression patterns: virus infected vs cancerous cells vs healthy cells

30
Q

Microarray Analysis process

A

1) DNA of cDNA from experimental sample deposited on glass slide alongside known DNA sequences
2) genomic DNA or mRNA can be isolated fro the two samples for comparison
- if mRNA isolated: it is reverse-transcribed to cDNA using reverse transcriptase
3) two samples of DNA or CDNA are labeled with different fluorescent dyes (red and green)
4) the DNA samples are combined in equal amounts and added to the microarray chip; the samples are allowed to hybridize to complementary spots on the microarray

31
Q

How is microarray analysis interpreted?

A

by measuring the intensity of fluorescence at particular spots on the microarray
-if both samples’ nucleic acids hybridize, then the spot will appear yellow due to combination of red and green dyes

32
Q

Downfalls of microarray analysis?

A

-requires expensive and sophisticated detection equipment and analysis software
-limited to research settings

33
Q

What are the two Amplification-Based DNA Analysis Methods?

A

Polymerase Chain Reaction & DNA Sequencing

34
Q

Polymerase Chain Reaction characteristics

A

permits rapid amplification in the number of copies of specific DNA sequences for further analysis
- in vitro lab techinque
-DNA primers used

35
Q

PCR occurs over multiple cycles containing three steps…

A

1) denaturation: 95C physically separates DNA strands
2) annealing: 50C allows target strand to stick to complementary strand
3) extension: 72C allows DNA polymerase to add nucleotides to primer using single stranded target as template

36
Q

How many cycles does PCR undergo?

A

25-40 cycles

37
Q

Reverse transcriptase PCR is used for…

A

obtaining DNA copies of a specific mRNA molecule

38
Q

Reverse transcriptase PCR process

A

1) reverse transcriptase enzyme converts mRNA molecules into cDNA
2) cDNA is used as a template for traditional PCR amplification
3) can detect whether a specific gene has been expressed in a sample

39
Q

real-time PCR (quantitative PCR) why is it quantitative?

A

the use of fluorescence allows one to monitor the increase in a double-stranded template during a PCR reaction as it occurs
-can determine amount of original target sequence

40
Q

Why are standard PCR and RT-PCR not quantitative?

A

because any one of the reagents may become limiting before all of the cycles within the protocol are complete, and samples are only analyzed at the end
- not possible to determine when a reagent has become limiting
- not possible to determine how many original molecules were present in the sample at the start of PCR

41
Q

qPCR is beneficial for…

A
  • determine the number of DNA copies
  • determine viral load in HIV-positive patients to evaluate effectiveness of their therapy
42
Q

Sanger DNA sequencing (dideoxy method) is

A

DNA replication of a single-stranded template with the use of a DNA primer to initiate synthesis of a complementary strand, DNA polymerase, a mix of four dNTP monomers, and a small proportion of dideoxynucleotides (ddNTPs)
-each ddNTP labeled with different color fluorescent dye so all four can be sequenced at once
-each band color will reveal nucleotide sequence of template strand

43
Q

What are ddNTPs?

A

monomers missing a hydroxyl group at the site at which another nucleotide usually attaches to form a chain
-terminates the process of DNA replication for the particular strand

44
Q

Difference between PCR and Next Generation Sequencing (NGS)

A

PCR: good for amplifying one gene at a time
NGS: amplification of a whole genome (takes place on a flow cell surface)

45
Q

Next generation sequencing benefits

A

+ rapid sequencing
+ low-cost
+ generate millions of short fragments in just one day
+ sequence whole genomes

46
Q

genomics

A

the study and comparison of entire genomes, including the complete set of genes and their nucleotide sequence and organization

47
Q

transcriptomics

A

the science of the entire collection of mRNA molecules produced by cells
- compare uninfected and infected host cells
- understand pathogenic processes

48
Q

metagenomics or metatranscriptomics

A

allow study of genes and gene expression from a collection of multiple species
-DNA microarray can be used

49
Q

proteomics

A

study of the entire complement of proteins in an organism (proteome)
-compare protein expression patterns between different organisms
-biomarkers: proteins who can detect various forms of cancer as well as other infections

50
Q

reporter genes encode & example of common one

A

easily observable characteristics to genes of interest
-gives location of gene
ex) lacZ

51
Q

what are the non-coding RNA molecules involved in RNA interference?

A

antisense RNA molecules

52
Q

What are antisense RNA molecules?

A

molecules complementary to regions of specific mRNA molecules found in pro and eukaryotic cells

53
Q

what is RNA intereference?

A

natural regulatory mechanism by which mRNA molecules are prevented from guiding the synthesis of proteins
-antisense molecules base pair to regions within complementary mRNA molecules, preventing protein synthesis

54
Q

Benefits of RNAi?

A

cells protected themselves from viral invasion
-researchers trying to develop techniques to mimic natural process of RNA interference as a way to treat viral infections in eukaryotes

55
Q

Shuttle Vectors & common example

A

plasmids that can move between bacterial and eukaryotic cells
ex) Agrobacterium – contain Tumor inducing plasmids that are commonly used as shuttle vectors for incorporating genes into plants

56
Q

Agrobacterium Ti plasmid shuttle vector process

A

isolate plasmid –> isolate gene of interest from cellular DNA –> clone gene of interest into Ti plasmid (making it recombinant) –> transform recombinant DNA back to bacteria –> infect plant cell with bacteria –> results in a recombinant plant

57
Q

T-DNA region composed of…

A

Auxin, Cytokinin, Opine

General Microbiology (3 decks)

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