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Gene Analysis
Biochem and medical genetics
| Question | Answer |
|---|---|
| Molecular biology methods | Hybridisation based techniques - nucleic acid detection Enzyme based techniques - manipulation of nucleic acids Use of antibodies - protein detection |
| Use of complementary probes - hybridisation | Double stranded DNA denatured to separate strands Flooded with smaller, tagged nucleic acids - probes Due to their high conc on cooling these probes bind to their complementary DNA You can then visualise the complementary DNA |
| Fluorescence In Situ Hybridisation - FISH | Cells fixed and introduced to a labelled probe The cells are treated with a denaturing agent and primers for a specific region The probe can then associate with the complementary sequence Fluorescence then allows that section of DNA to be identified |
| Chromosome painting | In situ approaches allow painting of a whole chromosome using specific labelled probes for DNA on that chromosome This allows for Spectral Karyotyping of whole chromosomes Can identify chromosomal abnormalities e.g. chromosomal rearrangements |
| RNA interference - miRNA | An important mechanism where cells use small 21 nucleotide long RNAs (miRNA) to regulate gene expression These are incorporated into the RNA silencing complex and act as guides to interact with a specific mRNA - can degrade or inhibit expression |
| RNA interference - siRNA | Delivering exogenous siRNAs complementary to a gene of interest allows control of that genes expression, studies of that gene and therapy |
| Example of siRNA therapy - Patisiran | A mutation in TTR can cause the protein (transthyretin in the liver) to adopt a different structure leading to amyloidosis as a result of aggregates in the heart and kidneys Patisiran is an siRNA targeting TTR mRNA, supressing its translation |
| CRISPR-CAS9 gene editing system | CRISPR associated CAS9 nuclease is a microbial adaptive immune system that has been repurposed for genome editing CAS9 nuclease can be introduced into cells and using guide RNA specifically directed to any location in the genome where it can cut DNA |
| How can CRISPR-CAS9 affect the genome | Mutations Specific sequence replacement/insertion Pairs of gRNA-directed CAS9 can stimulate large deletions or rearrangements gRNA directed CAS9 can fuse to activation domains to mediate regulation of genes, alter chromatin structure and enable imaging |
| Features of vectors used for cloning | Antibiotic resistance gene - selection of bacteria that take up the plasmid Origin of replication - replication independent of chromosomes Multiple cloning sites - unique restriction enzyme sites for cloning Phage promotors - enable transcription |
| Restriction enzymes | Natural endonucleases cutting DNA at specific recognition sequences Most widely used recognise 6 nucleotide palindromic sequences (read the same in both directions) GGATCC Leads to formation of sequence overhang |
| Ligation of DNA fragments | Two fragments digested by the same restriction enzyme can be joined together by a ligase enzyme Leads to covalent joining of separate DNA fragments |
| Recombinant DNA - Cloning | Cloning plasmid located in bacteria is isolated Cut by endonucleases and DNA inserted (cut by same enzyme) Forms recombinant DNA Bacteria then take these up - grown on agar containing ampicillin so only bacteria taking up the plasmid survive+ replicate |
| In vitro mRNA production | Capping - providing an artificial cap analogue Poly adenylation - transcribing a T-stretch on the template Polymerase terminated transcription by dropping off at the end of the linearized plasmid (by RE digestion) after the t stretch |
| Why introduce modified nucleotides in mRNA | Help reduce immunogenicity Stabilise transcripts E.g. methylated pseudo-uridines |
| Why use T7 RNA polymerase | Simple phage RNA polymerase that initiates transcription from o 18 nucleotide long promotor element without additional proteins - only nucleotides and a template |
| Polymerase Chain Reaction | Done in vitro Unlimited amplification of a chosen DNA fragment As DNA polymerases require primers, we can design oligonucleotides complementary to the sequence we want to amplify Done by cycling the temperature (95-55-72) |
| Real time PCR - SYBR green | Incorperation of dye into double stranded DNA results in fluorescence which is measured after each cycle Allows for an indication of relative abundance of a gene sequence The more DNA molecules present=the more fluorescence |
| Real time PCR - TaqMan | Oligonucleotide with a 5' fluorescent nucleotide and a 3' quencher is hybridised to the target sequence Polymerisation by tag releases the fluorescent nucleotide from the quencher - gives fluorescence as DNA is synthesised |
| Reverse transcription PCR | A retroviral derived polymerase reverse transcriptase is used to revers transcribe DNA from RNA templates Useful for analysis of mRNA as they all have a common 3'end - a short poly(t) primer can be used to form cDNA PCR can then be performed as normal |
| DNA sanger sequencing | Di-deoxynucleotides terminate polymerisation In addition to normal nucleotides the reaction mix also contains ddNTPs If polymerase inserts a ddNTP, the chain can't be extended as the last nucleotide lacks a 3' OH ddNTPs are labelled for identification |
| How is sanger sequencing analysed | Chain terminates at every possible position - each ddNTP nucleic acid ends a chain Separated by gel electrophoresis Laser beams are then passed through the gel, triggering the dye on ddNTPS to fluoresce, so nucleotide sequence can be read |
| Ion Torrent next-generation sequencing | DNA fragmented into small fragments and primers ligated onto each strand Each strand is placed in a well in a reader, and is then amplified As each nucleotide is passed over the wells, if it binds to a strand a proton is released - change in pH measured |
| How is ion-torrent read | Each mio read is mapped to the genome, allowing identification of its location Overlap of reads can be identified to see how they fit together Allows mutations to be identified |
| Single molecular sequencing platforms | Can read sequences of entire chromosomes When a chromosome is passed through a gap in the reader, each nucleotide changes the current produced in a specific way - allows rapid identification of the nucleotide |
| Immuno-florescence | Allows detection/localisation of a particular protein in a tissue Antibodies containing a fluorescent tag attached will bind to their specific protein E.g. identification of melanocortin 1 receptor by antibody that recognises its N-terminus |
| Immunohistochemistry | Use of antibodies which trigger a reaction in a tissue - a dye can be introduced that allows identification of that specific tissue E.g. identification of IHC status of breast cancer cells |
| Enzyme linked immuno sorbent assay | Capture antibody recognising a specific antigen is immobilised on surface of wells Sample containing the antigen is added After incubation the wells are washed, removing any non-captured material Detection antibody added - solution changes colour |
| Lateral flow test | Gold colloids conjugated with antibody for the antigen on conjugate pad Conjugated antibodies bind to target Immobilised antibodies trap Au-conjugated antibodies bound to antigen-test Immobilised antibodies bind free conjugated antibody-control |
| Western blotting | Exposure to SDS - uniform negative charge of proteins SDS-PAGE - fractionation of proteins Blotting to membrane Bath membrane is antibody solution for target protein Wash unbound antibodies away + add detection solution Expose to X-ray for detection |
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