TOPIC II: GENE TECHNOLOGIES Flashcards
What is a example of a giant virus?
The mimivirus- was thought to be a bacteria but is a virus 0.4-0.6 micrometers
- Pithovirus
- Pandoravirus
What has smaller genomes?
Parasitic lifestyles
- viruses are the ultimate parasite
What is the basic structure of a virus?
Protein coat with DNA or RNA
- inactive while extracellular
- inject genomic material in cells
What are RNA viruses?
Double or single stranded RNA genomes e.g. influenza, HIV, poliovirus
What are DNA viruses?
Double or SS DNA genome.
e. g. small pox, T4 bacteriophage
- GIant viruses are also DNA viruses
What is an example of a parasitic eukaryote?
Plasmodium
- contains an apicoplast (similar to choroplast)
- 3 genomes, 14 chromosomes
What contains two circular chromosomes?
Vibrio cholerae
What contains linear chromosomes?
Borrelia burguloferi-spirochaete
- Causative agent of lyme disease
What do plasmids encode?
- Non essential genes
- extrachromosomal
- circular or linear
What is a bacteriophage?
Virus that infects bacteria
What are transposable elements?
- Found within chromosomes- mobile DNA that can move in and out
e. g. insertion sequences, composite transposons, some bacteriophage
What are 4 facts regarding bacterial plasmids?
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
What is the plasmid copy number?
Average number of copies of plasmid in cell (1-100)
Why can it be good to have a high copy number in a plasmid?
Good if you want to overexpress a protein and amplify a gene
What are low copy numbers such as 1 plasmid good for?
Good for toxic things or if you want to have a genomic bank (more stable)
What are 5 functions of plasmids?
- control of replication (copy number control)
- Resistance (antibiotics and heavy metals)
- virulence genes (toxin production, secretion systems)
- Metabolic enzymes
- Production of antimicrobials (bacteriocins)
What is shigella?
E.coli with a virulence plasmid- 220kbp plasmid
- Without this plasmid shigella would NOT cause disease so it is a virulence plasmid
What is vertical transfer?
Vertical transfer (parent-->daughter) -normal reproduction - Related transfer
What is horizontal transfer and what are the three different types?
- 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)
What occurs in artificial transformation?
CaCl2 makes membrane more permeable
- Electroporation uses voltage to generate pores in membrane
What occurs in the horizontal transfer CONJUGATION INITIATION?
- 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)
What is a gene transfer experiment to do with conjugation?
- 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
What are the initial steps of conjugation?
- Pilus tip F+ binds to receptor on F- cell
- Pilus shrinks in length to make cells closer
- Conjugate bridge forms connecting cytoplasm of two cells
What are the DNA transfer steps in conjugation?
- 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
Is direct contact needed for transduction (horizontal transfer method) ?
NO! NO DIRECT CONTACT NEEDED!
What are the two cycles of transduction?
LYTIC CYCLE and the LYSOGENIC CYCLE
What occurs in the lytic cycle of transduction?
Phage injects DNA–> replicates–> lyses host cell and releases progeny phage
What is the lysogenic cycle of transduction?
- 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
What is GNEERALISED TRANSDUCTION?
- 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)
What is transfection and what are the two types? (form of horizontal transfer)
Introduction of foreign DNA into prokaryotic OR eukaryotic cells
Stable transfection and transient trasnfection
What is stable transfection?
Integrated into the chromosomes and more permanent
What is transient transfection?
Not permanent
- Expressed from a replicating vector/plasmid
What is a gene gun used for?
- To physically introduce DNA- DNA coupled to an inert solid fired directly into the nucleus
What is the process of a gene gun?
- 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
What is Agrobacterium tumefaciens?
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 )
How do we fix Agrobacterium tumefaciens in the lab?
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
What occurs in the GI phase?
Recruitment of the pre replication complex
What occurs in the S phase of cell cycle?
Activation of complex (kinases add phosphate to proteins-helicase is active)
What occurs in the detection of a gene?
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)
What does the amount of labelling (dark spaces) indicate in detection of a gene?
Amount of mRNA and therefore the expression level
What is PCR?
- DNA strands separate at high temperatures
- Heat removes helicase and ssBps (single stranded binding proteins)
- Provide primer by synthesis
What are the minimum requirements for amplification?
- tmeplate DNA
- DNA polymerase
- Primers (extremities of DNA template must be known )
- dNTpsv(deoxyribonucleotide triphosphate)
What is a problem faced in PCR?
Above the TM normal DNA polymerase is denatured and primers will not anneal
How do we solve the problem faced in PCR?
Temperature cycling. Increase temp to bind DNA and decrease temp to bind primers and for optimum DNA pol activity
What is an advantage of Taq and a disadvantage?
Pro: 20 cycles without inactivation
Con: Lacks proofreading
How does a PCR machine work?
Using a heating bloc that creates a gradient of temperature
What are the main uses of PCR?
- cloning purposes (replicate gene of interest)
- Change sequence (by changing primer)
- cDNA clones
What does qPCR do?
Quantifies DNA
Where can PCR be used?
- 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
How does the application of PCR work in forensic science?
- 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
What are some more unusual applications of PCR?
- 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
When do the levels of mRNA differ?
- 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)
What is the difference between a genomic library and cDNA library?
- 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)
Can we make a cDNA library in prokaryotes?
NO! Because mRNA is very unstable and doesn’t contain post modification steps
What things does the genomic library contain?
- Promoters
- Introns
- Intergenic
- NON EXPRESSED GENES
- Satellite DNA
What does the cDNA library contain?
- EXPRESSED GENES
- Transcription start sites
- Open reading frames (no gaps between exons)
- Splice points
What is satellite DNA?
Tandemly repeating ,non coding DNA
What is the preferred method of making genomic library?
F plasmids (Bacterial Artificial Chromosomes, BAC) -These are present only in one or two copies in E coli so they are stable
How are cDNA libraries made?
- 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)
What are the differences between Sanger sequencing and NGS?
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.
What is the detail of the sanger method?
- 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
What is the result of the Sanger method?
- Reaction mixture with ddATP will produce set of DNAs of different lengths terminating at each of the different As
In Sanger sequencing, how can the length of the strand be determined?
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
What are the modifications to the original Sanger sequencing method?
- 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
What is primer walking (application of sequencing in the lab)?
- 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)
What are some applications of sequencing in the lab?
- 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
What is shotgun sequencing?
- 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)
How do we determine the function of genes?
- genetics/biochem (mutation analysis)
- Comparative genomics (relationships within fmaily of genes -sequence homology)
Which technique can be used to identify the location of rpoB gene within S aureus?
WHOLE genome sequencing (not single) and molecular probes/fluorescent in situ hybridisation
Which technique can be used to determine if either of the S.aureus strains harbour any other known resistance genes?
If you know what you are looking for- PCR (quick answer)
- Microarray is same but EXPENSIVE
Which technique can be used to detect the presence of rifampicin resistant S.aureus in other tissues?
Molecular probing/ fluorescent in situ hybridisation is best but technically all methods can be used
Which technique can be used to investigate the function of the protein that rpoB encodes?
Cloning and expression of recombinant protein
