Analysis of the genome

• Transcriptome - The RNA content of a given cell at a given time

• Nanopore sequencing is where you denature the DNA using a helicase, and then pass it through a pore, changing the conductance.
• Every base gives a unique change in potential peak. 

• These are RNAs that the genome produces that don't produce a protein.
• They are difficult to predict from the genome sequence.
• LncRNA is found overlapping with protein coding genes.
• lincRNA is intergenic; it does not overalp with protein coding genes.

• PANDA is a lncRNA that is coded for by a stretch of DNA that acts as an enhancer region for a gene.
• Therefore, the PANDA acts as a sink, preferentially binding the transcription factor NF-YA and sequestering it. 

• A Microarray is a dish with wells containing known gene fragments.
• One sample transcriptome is labelled red, the other green.
• The colour the well goes upon RNA sample addition shows whther the gene is present in the transcriptome (yellow means it is present in both).
• As one gene can have multiple transcripts (differential splicing) it is possible that matches can be missed.

1. RNA broken into fragments and converted to c-DNA.

2. The DNAs are sequenced and assembled.

(3. A computer can detect different splice variants).

• RNA seq allows you to quantify amounts of RNA and to compare the levels of expression.
• It can also be used to predict susceptibility to chemotherapy drugs.

• This is the go to method for sequencing the proteome
• An antibody is attached to the dish via a protein.
• If the complementary protein is present in the sample, it will bind to the sample, and a detection antibody can then be added with a flourescent marker to see if there is a positive result.

• Another way to sequence the proteome

1. Load protein sample.

2. Separate by charge - the more charge the protein has the further it will move.

3. Rotate the gel.

4. Separate by molecular weight.

2D difference gel electrophoresis (2D DIGE)

• A dye for two investigated proteins are added to the sample
• A 2D gel is run
• If the proteins are expressed in equal amounts, there will be a yellow spot, with one or the other being dominant creating a green or red spot.

• This is a good way of detecting post-translational modifications
•  Protein is vaporised and separation depends on the mass:charge ratio

1. Melt the DNA strand at 94 degrees

2. Anneal the primers at 40-60 degrees

3. Extend the primers at 72 degrees.

• Two repeats give the desired fragment becasue the DNA polymerase will keep extending beyond the primers the first time, it is only when the DNA polymerase does a second round that the correct length is found (because the point where it started is already the right length).

• The DNA polymerase most commonly used is taq polymerase, because it does not denature at 94 degrees.
• As taq is highly error prone, another option is pfu, but it is less productive, so mixtures are usually the way to go.

1. Confirming the presence of a DNA sequence

To show that the primers didn't bind randomly you use nested PCR, which is where you use an outer and inner set of primers to gradually reduce the sequence to the desired one.

2. Characterising length polymorphisms

More commonly known as DNA fingerprinting.

3. Isolation of polymorphic alleles

4. Cloning DNA sequences

DNA can be inserted into plasmids using blunt ends if you introduce restriction sites into primers.

• This is used to convert RNA into DNA

1. A polyT primer (attaches to the polyA tail) is used to synthesie the cDNA using reverse transcriptase (gene specific primers can also be used).

2. There are two ways to make the cDNA double stranded and amplify it

                i. Use a thermostable DNA polymerase and gene specific primers. (the Tth thermostable                     DNA polymerase also has reverse transcriptase activity).

               ii. Add guanines to the end using a terminal transferase, and then add a polyC primer.

• A problem with normal PCR is that the amount of product formed is not proportional to the starting template, meaning that you can't really use it to find out how much starting material there is.

• To make it quantitative, you do this:

1. Light cycle assay - A dye (SYBR Green) is bound to the double stranded DNA, and as the DNA is amplified, there        will be more flourescence. 

2. Taqman assay - A probe is added that has a fluorophore and a condenser. As the DNA is amplified, the                 fluorophore is displaced from the quencher, allowing light to be emmited, The specificity of the probe means that you are measuring gene specific amplification. 

3. Molecular beacon - A quencher and reporter are added, and amplification moves the reporter away, allowing             fluroescence to occur. Info forms amplification plot.

1. Extract DNA from a mould

2. Break the DNA into fragments

3. Ligate the fragment into a cloning vector

4. Create a library in E. Coli

5. Identify the correct gene

6. Transfer to an expression/shuttle vector

7. Express in a cat

Cloning vector - An autonomously replicating piece of DNA that allows another piece of DNA to be amplified and manipulated.

Plasmids as cloning vectors

• pBR 322 - Has Ori, Amp, and Tet regions
• pUC18 - Has Ori, Amp, Lac Z, and Laz I regions

Physical methods

• Shearing - This is where the DNA is sucked up and down through a thin needle.
• Sonication - This is where a vibrating probe is placed in the DNA.

Enzymatic methods

• You can use different types of cutters

                        8 cutters - cut every 65kb

                        6 cutters - cut every 4kb

                        4 cutters - cut every 250bp

• At first glance the 6 cutter looks better as genes are about 1kb long adn you ideally want the whole gene, but 4 cutter with partial digest is best as 6 cutters have a high probability of cutting the middle of a gene

• Ligation uses the enzyme T4 DNA ligase and ATP, which adds and AMP to the phosphate groups at the ends.
• A nucleophilic attack by OH onto the P forms a phosphodiester bond.

1. Treat the cells with CaCl2 to increase their permeability

2. Mix the cells with the DNA and pass and electric current through in a process called electroporation.

3. Spread the cells on antibiotic-containing agar

• Each colony has a plasmid with has a section of the genomic DNA
• Bigger genomes require more clones:
•                 N = [ln(1-p)] / [ln(1-i/g)]
•                 p = probability of getting gene  //  i = average insert size  //  g = genome size

• If the organism has a lot of non coding DNA, then most clones will be useless

RNA libraries

• The solution to this is using an mRNA library.
• You can't clone RNA directly, as you must use reverse transcriptase to convert it to DNA before cloning

1. Use an oligo-dT primer and reverse transcriptase to add dTNPS, making the dirst DNA strand.

2. Methods for second strand synthesis

             i. Degrade the RNA with alkali and then add S1 nuclease (degrades the hairpin site that                    forms upon first strand synthesis), DNA polymerase, and dNTPs

            i. Digest the mRNA with RNAseH, and then add DNA polymerase and dNTPs using an                       RNA fragment as a primer.

            iii. Degrade the RNA and add a homopolymer tail to the 3' end using terminal                                      transferase. Use an oligo-dG primer, dNTPs and DNA polymerase to synthesise the                    second strand. 

• A cDNA clone does not have introns or promoters.

• To do this you use southern blotting.

1. Transfer the fragments onto a membrane by applying current.

2. Lyse and denature the fragments.

3. Hybridise them with a probe.

4. Identify the correct clone. 

• The probes used in southern blotting can be RNA, ssDNA, denatured dsDNA, or simply oligonucleotides.
• They must be labelled with a radioactive, flourescent, or ligand marker.
• Only the amino acid sequence is usually known though, and one amino acid can have many different codons...

Must have:

• Promoter
• A multiple cloning site
• A  terminator region
• An origin of replication
• An antibiotic resistance gene

• The gene must be cloned in the correct region with resepct to the histidine tag. This is indicated by the NDE1 site, which includes the start codon.

Mammals

• Mammals need shuttle vectors, that has parts that allow it to work for humans
• Flag tags can be added to them, which are used to study protein-protein interactions

• These are viral genes that have been altered (remove gag pol env and replace with transgene), that enter the cell but do not replicate.