BIO 230 Lab 3

Prokaryotic rRNA

• Has about 7 copies of the gene coding for this RNA

• make about 15,000 ribosomes per cell

Eukaryotic rRNA

• has about 50 to 5000 sets of genes coding for these rRNAs

• can have 10 million ribosomes at one time

How to isolate rRNA

Through sedimentaiton using a sucrose gradient where the rate of sedimentation is measured in Svendberg units or by gel electrophoresis

Higher concentration of agrose

Used to better resolve small fragments of DNA

Low concentration of agrose

Use to better resolve long fragements of DNA

Factors that influence rate of migration in gel electrophoresis

Amount of agarose used and the molecular structure (ex. mass to charge ratio - more negatively charged move faster towards the cathode)

Pulsed field gel electrophoresis

• Form of agarose gel electrophoresis that helps to separate long DNA molecuels

• Most electrophoresis has a consistent electric field - extends the molecule and allows it to migrate at a rate that is not dependent on length

• However this technique the electric field will changes the direction every so often

• Larger moleucles need more time to reorient in the changing field, therefore they migrate slower

• This can allow for bacterial or yeast chormosomes to be seaprated

• althought most mammals chromosomes are too large to be separted this way it can help to separate segments of these chromosomes if they are already cut with restriction enzymes

Ethidium bromide

• A dye that binds to DNS and gives off flourescence under UV

• May use a radioisotope or chemical markers to do this

Radioisotope

A modified DNA molecule with a molecule that creates light when that bases pairs to the DNA with the help of DNA polymerase, which allows bands of DNA to be visualized after gel electrophoresis

Sodium dodecyl sulfate polyacrylamide-gel electrophoresis

• Contains a highly cross linked polyacrylamide gel as migration matrix for protein

• Made by polymerizing monomers to form pores and the concentration of the gel can be varied to create pores that restrict the movement of proteins of a certain size

• Proteins dissolved in a highly negatively charged detergent

• Detergen binds to the hydrophobic regions of proteins allowing them to unfold and extend into polypeptide chains

• Beta mercaptoethanol is used to break disulfide bonds

• Proteins only separated by length as net charge is masked by the anions binding throughout the protiens

• has a large electric affinity to migrate to cathode

• Same size proteins go through at the same rate becasue they have the same length and will bind similar amounts of SDS

• Larger proteins have more charge have larger electrical forces but have harger drag

• caused large molecular proteins to be held back more

• Major protiens are detected by staining with gel in the dye like Coomassie blue

• smalller protien require a silver stain

• may also used a radioactive isotopte tag

Illumina sequencing

• Named after company that makes it - considred a second generation sequencing technique

• Create libraries of small DNA fragments that represent the DNA of a genome thorugh PCR amplification of many DNA fragments

• These DNA molecules are attached to the class surface of a flow cell and are bound to the original DNA fragment to keep them together - allowing 1000 copies of the gene to congregate together around this area of the genome

• Such copies fit into one flow cell and are sequenced in parallel

• Uses chain terminated nucleotides labeled with a fluorescent tag that can be removed

• Therefore when they are added - photo is taken of the reaction that occurs and records the identity of the colour and thus the specific nucleotide

• Coloured label is then remove and DNA polymerase can add the next nucleotide

• allows this to occur in parallel hundrends of times per cluster to sequence the section

• computer stitches together the sequences however this can be challenging to do

Single molecule real time sequencing

• Third generation sequencing technique

• occurs in small wells with DNA polymerase at the bottom surface

• As DNA polymerase copies the template DNA, fluorescent nucleotide is added that fluoresces a colour related to the identity of the nulceotide

• disapprers when the terminal phosphate is released in adding the phosphate into DNA chain, creating a flourescent pulse that is unique to each nucleotide

• Allows for th reading of long DNA fragments - 10s of thousands of reactions can be analyzed at a time

• Circular DNA can also be added to the ends of the dsDNA which then allows for the DNA fragment to be synthesized for a long time by DNA polymerase

Nanopore sequencing

• Third generation sequencing method

• allows for single stranded DNA to go through a protein pore in a membrane that has a voltage applied

• As each nucleotide passes through it creates a unique shift in the electric current to its identity- this is good as modified nucleotides can be detected as they will have slight changes in the way they impact the current (ex. 5-methylcytosine)

• quantifying these changes allows us to determine the identity of the DNA

• allows for long DNA to be sequenced and is poratble therefore it can be done in remote areas

• developing a direct RNA sequencing method for this

RNA seq

Currently involves the conversion of RNA into cDNA libraries that are analyzed through DNA sequencing although direct sequencing methods are being developed

CRISPR

• Uses guide RNA and cas9 protein to locate dsDNA to be cleaved

• helpful in creating transgenic organisms

• Cas 9 expressed in cultured cells witha guide RNA that is made to attack an area of the genome, complex will go and make cuts at specifi carea

• Breaks are repaired with nonhomologous end joining that creates errors in the sequence that stop gene function and inactivates the gene

• Most effective it is indues a framshft mutation near the start of the coding sequence

• to have a more precise gene knock out or replacement , Cas 9 and the guide RNA can be co-expressed in cells with a homologous gene sequence that codes for the correct gene that allows for homologous recombination to be used to insert the proper DNA sequence

• allows the gene to be selectively damaged and replaced a high efficiency

• Can be used to turn off a gene by creating a complementary sequence to a transcription activator or repressor and fusing it to a Cas9 protein which can essentially target any sequence in the gemome

Advantages of CRISPR

1. since it is based on the idea of complementary base pairing if you know the sequence of interest you can easily get the complementary guide RNA

2. do not have to modifiy the gene itself - works with DNA sequences already in the genome

3. many genes controlled at once - cas 9 only has to be made once but other guide RNAs can be expressed in the same cell = therefore a whole set of genes can be turned on or off