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BB10006 Cell and Molecular Biology > DNA modifications/Genetic Engineering > Flashcards

DNA modifications/Genetic Engineering Flashcards

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1
Q

What does methylated DNA cause?

A

Reduced transcription due to condensing of chromosomes

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2
Q

What DNA modification includes the methylation of a base? Which base is it and what product is formed?

A

Cytosine—>5-methylcytosine

Does this via SAM-CH2 converting to SAM

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3
Q

Where does methylation of cytosine often occur?

A

On a CG di nucleotide

Or may happen on a gene that’s being inhibited

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4
Q

Is methylation of cytosine heritable?

A

Yes

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5
Q

What is hemi methylation?

A

Only one strand is methylated (of DNA)

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6
Q

What is De Novo methylation?

A
  • Preserving DNA methylation in mitosis

- DNMT1 is the proposed maintenance methyltransferase

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7
Q

What is the role of DNTMA/B in De Novo methylation?

A
  • they’re methyltrasnferases that set up DNA methylation in early development
  • methylation would occur at blastocyst stage
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8
Q

Where is 5-Hydroxymethylcytosine found?

A

In purkinje neurons and the brain

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9
Q

What is the mechanism/what’s involved in the the reaction where 5-methylcytosine—>5-hydroxymethylcytosine?

A
  • TET enzyme

- MeCP2 binds to 5HMC (promote chromatin condensation)

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10
Q

What do mutations in MeCP2 gene cause?

A

Rett syndrome (an inherited neurodevelopmental disorder)

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11
Q

What technique would you use to map 5MC and 5HMC in a genome?

A
  • standard sequencing by synthesis approaches mean both are lost as they’re read as unmodified cytosines by DNA polymerase
  • next generation sequencing used as it enables efficient detection
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12
Q

What is Bisulphite sequencing?

A
  • chemically treat DNA molecules with Bisulphite so that ALL C’s —> U’s
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13
Q

What happens to methylated C’s in Bisulphite sequencing and what can this determine?

A
  • they’re not converted to Uracils

- can therefore determine positions of methylated C’s in original DNA molecule

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14
Q

What is a draw back of Bisulphite sequencing?

A

Can’t distinguish between 5MC or 5HMC

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15
Q

What is the method which Bisulphite sequencing is performed by?

A

Sanger (targeted) or Illumina (screening) sequence

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16
Q

What is TAB sequencing?

A
  • automisation of Bisulphite sequencing to enable 5HMC sites to be identified
  • methods run in parallel with BS sequencing
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17
Q

What does TAB need to work?

A

Glucosyl-transferase and TET enzyme

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18
Q

What is DNA sequencing by synthesis?

A
  • primer binds to DNA sequence you want to sequence then polymerase binds and you get a clone of sequence
  • most effective way to sequence DNA
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19
Q

What is Sanger sequencing?

A
  • DNA clones generated by standard PCR reaction
  • Clones then sequenced during polymerase mediated synthesis
  • random termination of extension at each nucleotide position is critical
  • ddNTPs added , read colours up gel to determine sequence
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20
Q

What does random termination of extension at each nucleotide position in Sanger sequencing result in?

A
  • DNA fragments of varying sizes which can be analysed to determine the nucleotide sequence
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21
Q

What is added to Sanger sequencing?

A

ddNTPs are added which differ from dNTPs.

  • they terminate the sequence when run on polyacrylamide gel
  • ddNTPs are labeled florescently
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22
Q

How do you determine the sequence when using Sanger Sequencing?

A
  • read the colours up the gel

- amplify signal in an electropherogram and order of colours gives sequence

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23
Q

What is Illumina sequencing?

A
  • next generation sequencing platform
  • localised Bridge application on glass slide to yield clonal clusters of DNA
  • reversible dye terminator chemistry to then determine DNA sequence of clonal clusters
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24
Q

What occurs in Clonal bridge amplification in Illumina sequencing?

A
  • Synthesised DNA stuck to glass slide
  • glass slide is a flow cell (has primers attached)
  • primer like adapters Added to both ends which enable the attachment of fragments permanently to the slide
  • Slide is bathed in a solution containing buffer polymerase then extension occurs
  • end Up with copy and original both attached to the glass on a single point
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25
Q

Pros and cons of Clonal Bridge amplification in illumina sequencing

A
  • Quickly sequence whole gene aims in a short time but not as accurate
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26
Q

What occurs in reversible dye terminator chemistry in illumina sequencing?

A
  • four types of reversible dye terminator which added (ATCG)
  • Each nucleotide is Fluorescently labelled
  • 4 nucleotides compete for binding sites on the template DNA
  • A laser is applied to remove blocking group
  • The colours become visible from each fluorescent nucleotide which allows for the sequence identification of the entire DNA sequence
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27
Q

What does single molecule real time sequencing enable? SMRT

A
  • Enables DNA to be sequenced without PCR amplification

- Enables direct Detection of DNA modifications

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28
Q

What occurs in single molecule real time sequencing?

A
  • Plate in wells, polymerase attached at the bottom of well
  • Illuminate that part of the well - zero mode waveguide
  • Four bases have fluorescent dye , when the nucleotide is incorporated by polymerase the fluorescent tag is cleaved off
  • Detector detector dye and the base is determined
29
Q

In single molecule real-time sequencing what does a delay suggest?

A

A modification

30
Q

What do transient transfections allow?

A
  • Short term examinations to be conducted

- The inner compartments are aqueous

31
Q

What do stable transfections enable?

A

Longer term investigations

32
Q

Steps of a transient transfection?

A
  • Fusogenic lysosome combines with genetic material (RNA)
  • Fuses with cell membrane
  • Release of RNA in the cytoplasm
33
Q

What is the GFP protein and what Is it used for?

A
  • Used to follow/track location of targeted protein/genes
  • found in jellyfish
  • insert GFP into vector downstream of promoter it will then be expressed
  • Insert multiple cloning site downstream of promoter
  • The gene of interest will be expressed as fusion protein with GFP
34
Q

What enzymes are involved in a stable transfection of mammalian cell lines done via viral vectors and virus mediated infection?

A
  • Reverse transcriptase converts RNA genome to DNA
  • Intergase insert DNA molecule into genome
  • more laborious
35
Q

What is a packaging cell?

A

Used to make a vector.
Vector engineered to have a signal to trick the virus into thinking it’s the genome.

Eg. Retroviruses

36
Q

What is a transgenic model of human disease?

A
  • Diseases caused by mutation in a single gene
  • can generate transgenic models
  • different types of models have different advantages
  • Place selection marker inserted inside your gene, transform into a mouse cell, get homologous recombination
  • knock out a gene
37
Q

What is homologous recombination?

A

Nucleotide sequences exchanged can repair damaged DNA

38
Q

What occurs within a targeting vector in a mouse?

A
  • Take early stage embryo (blastocyst) and take cells from it
  • homologous recombination occurs
  • Select cells which have undergone recombination and apply meomisin
  • only cells which express the gene will survive
  • Take these and inject into another cell from mouse then transfer to a surrogate mother
  • cross with a white mouse you’ll get a black mouse
39
Q

What is the Cre-LoxP based methodology?

A
  • Desirable to have gene deleted only in specific tissue/organ of interest
  • can engineer the loxP site to generate mouse containing floxed allele
  • Cross with mouse containing Cre recombinase gene, then you get expression in heart
40
Q

What is haemophilia A caused by?

A

A mutation in the gene encoding factor VIII which is required for normal blood clotting

41
Q

Where is the factor eight gene located?

A

On the X chromosome

42
Q

What is required to stop abnormal bleeding?

A

The factor eight gene

- replacement therapy used to stop bleeding

43
Q

Stages of curing haemophilia A

A
  • Recombinant factor eight produced in hamster cells
  • Now there’s mammalian cells in which stable transactions can be performed
  • Human cell line recently engineered (HEK293-F) amenable to stable transfection
  • this has been used to produce factor eight on an industrial level
44
Q

What is the issue with using factor eight produced in hamster cells in human cells?

A

Because of post-translational modifications when the human protein produced in hamsters unnatural molecules are added causing in immunogenic reaction

45
Q

What are the two types of gel used in electrophoresis?

A

Agarose and polyacrylamide

46
Q

What occurs in electrophoresis?

A

Gel mesh, place molecules in gel mesh, apply potential difference across and molecules will move

47
Q

How would you prepare an agarose gel?

A

Mix with buffer, microwave, melts, comb to make wells, load sample onto it
-can mix dye with sample then visually track it

48
Q

What is agarose gel electrophoresis good for?

A

Medium-size nucleic acid analysis

49
Q

What is polyacrylamide gel electrophoresis suited for?

A

Smaller nucleic acid analysis as it has better resolving power

50
Q

What Are the stages in polyacrylamide gel electrophoresis?

A

Create a small gap then pour into glass plates

- Oxidation prevents cross-linking reaction so it’s essential you overlay with water to prevent this

51
Q

What stains can be used in PAG?

A
  • ethidium bromide, not safe as other stains but cheap
  • sybr gold is safer but more Expensive
  • coomassie blue is good for protein stain
  • Use transilluminator for visualisation of nucleic acids
52
Q

What do denaturing conditions in electrophoresis involve?

denaturing vs native electrophoresis

A

Chemically treating nucleic acids

- eg with formamide / SDS (reducing agent)

53
Q

What is southern blotting?

A
  • Enables the determination of presence of DNA species of interest within a sample
  • Invented in 1975 by Ed southern
  • Being largely suspended by newer methods e.g. PCR
  • DNA fragments separated by size and charge during electrophoresis
  • DNA fragments transferred on nylon membrane, then Desired DNA detected using DNA probe that is complimentary to desired DNA
  • Hybridisation probe
54
Q

What is northern blotting?

A
  • Same but RNA
  • Quick and easy way to determine expression pattern of a gene of interest across tissues/organs
  • RNA Extracted, mRNA Isolated, then is formamide denatured and run on PAGE gel for blotting
  • Radioactively labelled probe complementary to RNA
55
Q

What Differences are there in western blotting?

A
  • Ab raised against protein of interest is used as the probe
  • proteins are usually electrophoretically transferred onto nitrocellulose membrane
  • Denaturing conditions
  • Protein separated on size only
56
Q

What is pulse field electrophoresis?

A

Enables analysis of longer/larger DNA

57
Q

What is EMSA Electrophoresis?

A

DNA protein interactions analysed by this

-non-denaturing PAGE conditions

58
Q

What is SSCP electrophoresis?

A
  • non denaturing gels, used to detect mutations in DNA

- quick and robust no need for DNA sequencing

59
Q

What does a restriction endonuclease do?

A

Cut phosphodiester bonds e.g. ECOR1

60
Q

What do ligases do?

A

Catalyse formation of phosphodiester bonds between DNA ends

61
Q

What is restriction endonuclease produced by?

A

Bacteria to combat phage infection

  • DNA destroyed in bacteriophage, restriction enzyme doesn’t cut DNA as it’s methylated
  • ECORV methylase will methylated DNA seq
62
Q

What do plasmids often contain?

A

Antibiotic resistance gene

63
Q

What are expression vectors engineered to have?

A

The promoter adjacent to a multiple cloning site

64
Q

What is site directed mutagenesis?

A

Where are you introduce a mutation into a vector, the primer contains the mutation

65
Q

What is the Cre-LoxP system?

A
  • Study genetic function
  • in centres on the cyclisation recombinase enzyme
  • cre recognises specific sequences known as LoxP sequences and introduces recombination between them
66
Q

What is the Crispr-Cas9 system?

A

-Cellular system in Bacteria to combat viral infection

– could treat and kill genetic diseases

67
Q

Bacterial genomes are usually protected from the action of restriction endonucleases via which modification to their own DNA?

A

Methylation

68
Q

Liposomes used for cellular transfection of genetic material are composed of:

A

Phospholipids

69
Q

The 6-methyladenosione modification present in cellular mRNAs can be bound by the YTHDF2 protein which can lead to

A

Degradation of the mRNA

BB10006 Cell and Molecular Biology (5 decks)

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