DNA modifications/Genetic Engineering Flashcards
What does methylated DNA cause?
Reduced transcription due to condensing of chromosomes
What DNA modification includes the methylation of a base? Which base is it and what product is formed?
Cytosine—>5-methylcytosine
Does this via SAM-CH2 converting to SAM
Where does methylation of cytosine often occur?
On a CG di nucleotide
Or may happen on a gene that’s being inhibited
Is methylation of cytosine heritable?
Yes
What is hemi methylation?
Only one strand is methylated (of DNA)
What is De Novo methylation?
- Preserving DNA methylation in mitosis
- DNMT1 is the proposed maintenance methyltransferase
What is the role of DNTMA/B in De Novo methylation?
- they’re methyltrasnferases that set up DNA methylation in early development
- methylation would occur at blastocyst stage
Where is 5-Hydroxymethylcytosine found?
In purkinje neurons and the brain
What is the mechanism/what’s involved in the the reaction where 5-methylcytosine—>5-hydroxymethylcytosine?
- TET enzyme
- MeCP2 binds to 5HMC (promote chromatin condensation)
What do mutations in MeCP2 gene cause?
Rett syndrome (an inherited neurodevelopmental disorder)
What technique would you use to map 5MC and 5HMC in a genome?
- 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
What is Bisulphite sequencing?
- chemically treat DNA molecules with Bisulphite so that ALL C’s —> U’s
What happens to methylated C’s in Bisulphite sequencing and what can this determine?
- they’re not converted to Uracils
- can therefore determine positions of methylated C’s in original DNA molecule
What is a draw back of Bisulphite sequencing?
Can’t distinguish between 5MC or 5HMC
What is the method which Bisulphite sequencing is performed by?
Sanger (targeted) or Illumina (screening) sequence
What is TAB sequencing?
- automisation of Bisulphite sequencing to enable 5HMC sites to be identified
- methods run in parallel with BS sequencing
What does TAB need to work?
Glucosyl-transferase and TET enzyme
What is DNA sequencing by synthesis?
- 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
What is Sanger sequencing?
- 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
What does random termination of extension at each nucleotide position in Sanger sequencing result in?
- DNA fragments of varying sizes which can be analysed to determine the nucleotide sequence
What is added to Sanger sequencing?
ddNTPs are added which differ from dNTPs.
- they terminate the sequence when run on polyacrylamide gel
- ddNTPs are labeled florescently
How do you determine the sequence when using Sanger Sequencing?
- read the colours up the gel
- amplify signal in an electropherogram and order of colours gives sequence
What is Illumina sequencing?
- 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
What occurs in Clonal bridge amplification in Illumina sequencing?
- 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
Pros and cons of Clonal Bridge amplification in illumina sequencing
- Quickly sequence whole gene aims in a short time but not as accurate
What occurs in reversible dye terminator chemistry in illumina sequencing?
- 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
What does single molecule real time sequencing enable? SMRT
- Enables DNA to be sequenced without PCR amplification
- Enables direct Detection of DNA modifications
What occurs in single molecule real time sequencing?
- 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
In single molecule real-time sequencing what does a delay suggest?
A modification
What do transient transfections allow?
- Short term examinations to be conducted
- The inner compartments are aqueous
What do stable transfections enable?
Longer term investigations
Steps of a transient transfection?
- Fusogenic lysosome combines with genetic material (RNA)
- Fuses with cell membrane
- Release of RNA in the cytoplasm
What is the GFP protein and what Is it used for?
- 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
What enzymes are involved in a stable transfection of mammalian cell lines done via viral vectors and virus mediated infection?
- Reverse transcriptase converts RNA genome to DNA
- Intergase insert DNA molecule into genome
- more laborious
What is a packaging cell?
Used to make a vector.
Vector engineered to have a signal to trick the virus into thinking it’s the genome.
Eg. Retroviruses
What is a transgenic model of human disease?
- 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
What is homologous recombination?
Nucleotide sequences exchanged can repair damaged DNA
What occurs within a targeting vector in a mouse?
- 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
What is the Cre-LoxP based methodology?
- 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
What is haemophilia A caused by?
A mutation in the gene encoding factor VIII which is required for normal blood clotting
Where is the factor eight gene located?
On the X chromosome
What is required to stop abnormal bleeding?
The factor eight gene
- replacement therapy used to stop bleeding
Stages of curing haemophilia 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
What is the issue with using factor eight produced in hamster cells in human cells?
Because of post-translational modifications when the human protein produced in hamsters unnatural molecules are added causing in immunogenic reaction
What are the two types of gel used in electrophoresis?
Agarose and polyacrylamide
What occurs in electrophoresis?
Gel mesh, place molecules in gel mesh, apply potential difference across and molecules will move
How would you prepare an agarose gel?
Mix with buffer, microwave, melts, comb to make wells, load sample onto it
-can mix dye with sample then visually track it
What is agarose gel electrophoresis good for?
Medium-size nucleic acid analysis
What is polyacrylamide gel electrophoresis suited for?
Smaller nucleic acid analysis as it has better resolving power
What Are the stages in polyacrylamide gel electrophoresis?
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
What stains can be used in PAG?
- 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
What do denaturing conditions in electrophoresis involve?
denaturing vs native electrophoresis
Chemically treating nucleic acids
- eg with formamide / SDS (reducing agent)
What is southern blotting?
- 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
What is northern blotting?
- 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
What Differences are there in western blotting?
- 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
What is pulse field electrophoresis?
Enables analysis of longer/larger DNA
What is EMSA Electrophoresis?
DNA protein interactions analysed by this
-non-denaturing PAGE conditions
What is SSCP electrophoresis?
- non denaturing gels, used to detect mutations in DNA
- quick and robust no need for DNA sequencing
What does a restriction endonuclease do?
Cut phosphodiester bonds e.g. ECOR1
What do ligases do?
Catalyse formation of phosphodiester bonds between DNA ends
What is restriction endonuclease produced by?
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
What do plasmids often contain?
Antibiotic resistance gene
What are expression vectors engineered to have?
The promoter adjacent to a multiple cloning site
What is site directed mutagenesis?
Where are you introduce a mutation into a vector, the primer contains the mutation
What is the Cre-LoxP system?
- Study genetic function
- in centres on the cyclisation recombinase enzyme
- cre recognises specific sequences known as LoxP sequences and introduces recombination between them
What is the Crispr-Cas9 system?
-Cellular system in Bacteria to combat viral infection
– could treat and kill genetic diseases
Bacterial genomes are usually protected from the action of restriction endonucleases via which modification to their own DNA?
Methylation
Liposomes used for cellular transfection of genetic material are composed of:
Phospholipids
The 6-methyladenosione modification present in cellular mRNAs can be bound by the YTHDF2 protein which can lead to
Degradation of the mRNA
