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BMS1062 BIO > TOPIC II: GENE TECHNOLOGIES > Flashcards

TOPIC II: GENE TECHNOLOGIES Flashcards

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Biology 101 flashcards
1
Q

What is a example of a giant virus?

A

The mimivirus- was thought to be a bacteria but is a virus 0.4-0.6 micrometers

  • Pithovirus
  • Pandoravirus
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2
Q

What has smaller genomes?

A

Parasitic lifestyles

- viruses are the ultimate parasite

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3
Q

What is the basic structure of a virus?

A

Protein coat with DNA or RNA

  • inactive while extracellular
  • inject genomic material in cells
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4
Q

What are RNA viruses?

A

Double or single stranded RNA genomes e.g. influenza, HIV, poliovirus

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5
Q

What are DNA viruses?

A

Double or SS DNA genome.

e. g. small pox, T4 bacteriophage
- GIant viruses are also DNA viruses

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6
Q

What is an example of a parasitic eukaryote?

A

Plasmodium

  • contains an apicoplast (similar to choroplast)
  • 3 genomes, 14 chromosomes
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7
Q

What contains two circular chromosomes?

A

Vibrio cholerae

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8
Q

What contains linear chromosomes?

A

Borrelia burguloferi-spirochaete

- Causative agent of lyme disease

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9
Q

What do plasmids encode?

A
  • Non essential genes
  • extrachromosomal
  • circular or linear
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10
Q

What is a bacteriophage?

A

Virus that infects bacteria

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11
Q

What are transposable elements?

A
  • Found within chromosomes- mobile DNA that can move in and out
    e. g. insertion sequences, composite transposons, some bacteriophage
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12
Q

What are 4 facts regarding bacterial plasmids?

A

Ds DNA, replcates by itself

  • Smaller than chromosomes
  • There are multiple copies per cell
  • They don’t encode functions essential for growth in all conditions
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13
Q

What is the plasmid copy number?

A

Average number of copies of plasmid in cell (1-100)

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14
Q

Why can it be good to have a high copy number in a plasmid?

A

Good if you want to overexpress a protein and amplify a gene

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15
Q

What are low copy numbers such as 1 plasmid good for?

A

Good for toxic things or if you want to have a genomic bank (more stable)

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16
Q

What are 5 functions of plasmids?

A
  • control of replication (copy number control)
  • Resistance (antibiotics and heavy metals)
  • virulence genes (toxin production, secretion systems)
  • Metabolic enzymes
  • Production of antimicrobials (bacteriocins)
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17
Q

What is shigella?

A

E.coli with a virulence plasmid- 220kbp plasmid

- Without this plasmid shigella would NOT cause disease so it is a virulence plasmid

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18
Q

What is vertical transfer?

A 
Vertical transfer (parent-->daughter) -normal reproduction 
- Related transfer
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19
Q

What is horizontal transfer and what are the three different types?

A
  • cell to UNRELATED cell
    1. Transformation (uptake of free DNA)
    2. Conjugation (direct transfer of DNA from cell-cell)
    3. Transduction ( transfer via a bacteriophage intermediate)
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20
Q

What occurs in artificial transformation?

A

CaCl2 makes membrane more permeable

- Electroporation uses voltage to generate pores in membrane

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21
Q

What occurs in the horizontal transfer CONJUGATION INITIATION?

A
  • bacterial DNA transferred by direct cell-cell contact
  • F plasmid involved
  • Must have DNA encoding a sex pilus
  • Express proteins for DNA transfer
  • DNA with region that can begin transfer (origin of transfer oriT)
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22
Q

What is a gene transfer experiment to do with conjugation?

A
  • Strain A bacteria can’t grow on medium because they need certain genes (that strain B has)
  • Strain B can’t grow on medium because it needs genes (that A has)
    = When mixed together, there is growth indicating DNA transfer
    MUST HAVE DIRECT CONTACT
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23
Q

What are the initial steps of conjugation?

A
  1. Pilus tip F+ binds to receptor on F- cell
  2. Pilus shrinks in length to make cells closer
  3. Conjugate bridge forms connecting cytoplasm of two cells
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24
Q

What are the DNA transfer steps in conjugation?

A
  • One strand of DNA nicked at the oriT site
  • One DNA strand pushed through conjugation bridge
  • F+ cell repairs plasmid
  • F- cell uses remaining DNA for template of DNA synthesis
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25
Q

Is direct contact needed for transduction (horizontal transfer method) ?

A

NO! NO DIRECT CONTACT NEEDED!

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26
Q

What are the two cycles of transduction?

A

LYTIC CYCLE and the LYSOGENIC CYCLE

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27
Q

What occurs in the lytic cycle of transduction?

A

Phage injects DNA–> replicates–> lyses host cell and releases progeny phage

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28
Q

What is the lysogenic cycle of transduction?

A
  • doesn’t always cause lysis
  • phage injects DNA and integrates into chromosome (NO LYSING, REPLICATION OR RELEASE OF PROGENY)
  • Phage DNA then replicated with host chromosome
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29
Q

What is GNEERALISED TRANSDUCTION?

A
  • Bacteriophage incorrectly packages bacterial DNA into the phage particle (must fit into phage head of <100kbp)
  • bacteriophage can still infect new bacteria but viable phage will not be produced from DNA ( can’t combine with chromosome)
30
Q

What is transfection and what are the two types? (form of horizontal transfer)

A

Introduction of foreign DNA into prokaryotic OR eukaryotic cells
Stable transfection and transient trasnfection

31
Q

What is stable transfection?

A

Integrated into the chromosomes and more permanent

32
Q

What is transient transfection?

A

Not permanent

- Expressed from a replicating vector/plasmid

33
Q

What is a gene gun used for?

A
  • To physically introduce DNA- DNA coupled to an inert solid fired directly into the nucleus
34
Q

What is the process of a gene gun?

A
  • plasmid coated with gene of interest (on tungsten or gold particles-microprojectiles)
  • Shoot- penetration of cell wall
  • Micro projectiles enter cells, transgenes may incorporate into chromosomal DNA
  • SELECTABLE MARKERS used to identify cells that make up trans gene
  • REGENERATE in plant cells
35
Q

What is Agrobacterium tumefaciens?

A

Causes crown-gall disease in plants

  • tumor like growth
  • insertion of small segment of DNA (T-DNA from Tumor Inducing plasmid)
  • T DNA integrated into the genome of host cell
  • T DNA carries genes for weird amino acids like octopine )
36
Q

How do we fix Agrobacterium tumefaciens in the lab?

A

Plasmid modified by substituting selectible marker gene and desired transgene between T-DNA repeats
- sequences between T-DNA repeats are efficiently transferred into the plant cell

37
Q

What occurs in the GI phase?

A

Recruitment of the pre replication complex

38
Q

What occurs in the S phase of cell cycle?

A

Activation of complex (kinases add phosphate to proteins-helicase is active)

39
Q

What occurs in the detection of a gene?

A

Probe (prepared ss DNA or ss RNA) labelled

  • Unknown sample (like purified DNA from cell)
    e. g. Amigo 2 in brain sections
  • To find out where in a particular region they are, the robes bind to mRNA strand (complementary base pairing)
40
Q

What does the amount of labelling (dark spaces) indicate in detection of a gene?

A

Amount of mRNA and therefore the expression level

41
Q

What is PCR?

A
  • DNA strands separate at high temperatures
  • Heat removes helicase and ssBps (single stranded binding proteins)
  • Provide primer by synthesis
42
Q

What are the minimum requirements for amplification?

A
  • tmeplate DNA
  • DNA polymerase
  • Primers (extremities of DNA template must be known )
  • dNTpsv(deoxyribonucleotide triphosphate)
43
Q

What is a problem faced in PCR?

A

Above the TM normal DNA polymerase is denatured and primers will not anneal

44
Q

How do we solve the problem faced in PCR?

A

Temperature cycling. Increase temp to bind DNA and decrease temp to bind primers and for optimum DNA pol activity

45
Q

What is an advantage of Taq and a disadvantage?

A

Pro: 20 cycles without inactivation
Con: Lacks proofreading

46
Q

How does a PCR machine work?

A

Using a heating bloc that creates a gradient of temperature

47
Q

What are the main uses of PCR?

A
  • cloning purposes (replicate gene of interest)
  • Change sequence (by changing primer)
  • cDNA clones
48
Q

What does qPCR do?

A

Quantifies DNA

49
Q

Where can PCR be used?

A
  • GENETIC TESTING e..g breast cancer (mutation in protein that doesn’t produce tumor sepressor proteins
  • PATERNITY TESTING (STR non coding 1-6 nucleotides-inherit from parents)
  • FORENSIC SCIENCE
50
Q

How does the application of PCR work in forensic science?

A
  • Uses VNTR (variable number of tandem repeats) (longer than STRs -100bp)
  • Individuals inherit each VNTR locus from mum and dad
  • Pair of primers that brackets VNTR loci provides bands of DIFFERENT sizes as inherited from each parent
  • Only needs TINY amount of material
51
Q

What are some more unusual applications of PCR?

A
  • eDNA (environmental) from waterways to detect Burmese python invading Florida evergrades
  • eDNA from excrement to detect invasive species (coconut beetle)
  • Monitor threatened species and genetic diversity
  • Finding out what killed certain animalse,e.g. coyote killing cat
  • Illegal meat
  • WIldlife poaching
52
Q

When do the levels of mRNA differ?

A
  • for different genes in the same cell
  • For same gene in different cell types or differentiation states
  • can differ in same gene in response to external signals, viral infection, insulin levels, oxidative stress (dictates phenotype of cell)
53
Q

What is the difference between a genomic library and cDNA library?

A
  • Genomic library contains NON expressed genes (introns and exons)
  • cDNA library contains only the parts that will be expressed and correspond to protein encoding genes (no introns present in cDNA library but contains spicing points)
54
Q

Can we make a cDNA library in prokaryotes?

A

NO! Because mRNA is very unstable and doesn’t contain post modification steps

55
Q

What things does the genomic library contain?

A
  • Promoters
  • Introns
  • Intergenic
  • NON EXPRESSED GENES
  • Satellite DNA
56
Q

What does the cDNA library contain?

A
  • EXPRESSED GENES
  • Transcription start sites
  • Open reading frames (no gaps between exons)
  • Splice points
57
Q

What is satellite DNA?

A

Tandemly repeating ,non coding DNA

58
Q

What is the preferred method of making genomic library?

A 
F plasmids (Bacterial Artificial Chromosomes, BAC) 
-These are present only in one or two copies in E coli so they are stable
59
Q

How are cDNA libraries made?

A
  • Extracting mRNA and reverse transcribing to make copy DNA
  • Poly T primer added to poly A tail to synthesise the complementary DNA
  • cDNA then converted into ds DNA with DNA polymerase
  • cDNA then injected into plasmid and cloned
  • entire collection of clones make the cDNA library
  • cDNA clones only contain regions of genome transcribed into mRNA (transcriptome)
60
Q

What are the differences between Sanger sequencing and NGS?

A

Sanger sequencing is convenient for relatively small number of samples (e.g. <96) while NGS is only efficient for analysing 100s or 1000s of genes.

61
Q

What is the detail of the sanger method?

A
  • uses modified nucleotides (removes a 3’ OH) which terminates the chain
  • DNA synthesised in mixture of ssDNA, DNA polymerase and 4 dNTPs
  • If dedioxyNTP analog of one of dNTP is present,can become incorperated into growing chain
  • can’t add next dNTP because of no 3’OH so DNA chain terminated at that point
62
Q

What is the result of the Sanger method?

A
  • Reaction mixture with ddATP will produce set of DNAs of different lengths terminating at each of the different As
63
Q

In Sanger sequencing, how can the length of the strand be determined?

A

By determining position of A in the growing chain

  • Repeated for ddCTP, ddGTP and ddTTP (each of the diff bases)
  • Newely synthesised sequence is COMPLEMENTARY to the template strand
64
Q

What are the modifications to the original Sanger sequencing method?

A
  • primer is not radioactive
  • 4 ddNTPs carry different FLUORESCENT COLOURED TAG each (instesd of 32P)
  • Reaction done in ONE tube (instead of 4)
  • Fragments separated in single lane
  • automatic laser scanning and recording
65
Q

What is primer walking (application of sequencing in the lab)?

A
  • First sequenced as a shorter fragment (sanger sequ.)
  • Use universal primers to identify the first 1000 bases
  • Design NEW primer complementary to final 20 bases of known sequence
    (keep ‘walking’ along sequence)
66
Q

What are some applications of sequencing in the lab?

A
  • To verify what you have made is what you though it was (make sure there are no base insertions, deletions, substitutions)
  • to verify NGS (next generation sequencing)
  • Primer bound in right place
67
Q

What is shotgun sequencing?

A
  • Chain termination (CTM) method only used fairly short strands (100-1000bp)
  • Longer sequence divided into smaller RANDOM fragments
  • CTM done
  • Multiple overlapping reads for target DNA are obtained over several rounds of fragmentation and sequencing
  • computer programs used to piece overlapping ends together into continuous sequence (full genome sequencing)
68
Q

How do we determine the function of genes?

A
  • genetics/biochem (mutation analysis)

- Comparative genomics (relationships within fmaily of genes -sequence homology)

69
Q

Which technique can be used to identify the location of rpoB gene within S aureus?

A

WHOLE genome sequencing (not single) and molecular probes/fluorescent in situ hybridisation

70
Q

Which technique can be used to determine if either of the S.aureus strains harbour any other known resistance genes?

A

If you know what you are looking for- PCR (quick answer)

- Microarray is same but EXPENSIVE

71
Q

Which technique can be used to detect the presence of rifampicin resistant S.aureus in other tissues?

A

Molecular probing/ fluorescent in situ hybridisation is best but technically all methods can be used

72
Q

Which technique can be used to investigate the function of the protein that rpoB encodes?

A

Cloning and expression of recombinant protein

BMS1062 BIO (7 decks)

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