Characterisation of genes and gene products at a molecular level Flashcards
DNA cloning definition
the process of making multiple identical copies of a particular piece of DNA.
Process of DNA cloning
- Restriction endonuclease cuts DNA at specific recognition sequence.
- Restriction endonuclease cuts complementary DNA at plasmid.
- DNA ligase joins the two fragments in the presence of calcium chloride which makes bacterial wall more permeable to the DNA fragment.
- DNA then multiplied rapidly in PCR, thus amplifying the DNA.
Special features of the recognition sequence
Often 6 bases long and produces a palindromic sequence.
What is a palindromic sequence?
Read the same 5’-3’ as 3’-5’
Features of cloning vectors
- Contain an antibiotic resistance gene for selection of the plasmids that contain the new gene.
- contains an origin of replication for the replication of the plasmid
- multiple cloning site
- phage promoters which enable in vitro transcription of the recombination DNA fragment
Explain DNA electrophoresis
electric field is established across a gel, causing the DNA molecules to move a certain distance dependent on their size
Why would DNA be different lengths?
Contain many recognition sites for different restriction endonucleases which results in the DNA being cleaved at different base pair lengths.
Stages of DNA electrophoresis
- Electric field established across the gel
- DNA will move to anode, as it is negatively charged due to phosphate groups
- Polymers forming the gels have a porous matrix, which acts as a sieve allowing shorter DNA to move more rapidly than larger ones.
- DNA chains are then visualised using different methods
What gel is used for electrophoresis?
polyacrylamide and agrose
Two different methods of visualising DNA
- Stain with ethidium bromide, a fluorescent compound that binds to DNA. Forms red-orange fluorescent bands under UV.
- Autoradiography- Radioisotope 32P is incorporated into DNA before electrophoresis, then placed on a radiographic film after migration is complete.
Hybridisation of nucleic acids process
- Double stranded DNA molecules denatured by heat
2. Complementary sequences re-hybridise at lower temperatures
3 processes based on DNA hybridisation
southern blotting, northern blotting, fluorescent in situ hybridisation (FISH)
Stages of Southern Blot
- DNA sample cleaved by a restriction endonuclease
- Placed onto a polyacrylamide and agrose gel and separated by electrophoresis
- Gel is immersed in sodium hydroxide to denature the DNA
- DNA transferred from gel onto another support, such as a sheet of nitrocellulose. This is done by adding layers of absorbent paper soaked in salt solution into a cuvette. Gel block containing DNA is placed over the paper block and the nitro-cellulose sheet is arranged at the top.
- Dry blotting paper is added to the top, causing the solution from the lower stack to rise, moving the DNA in the gel to the nitrocellulose.
- Nitrocellulose, now a replica of the DNA, is heated in an oven to fix and immobilise the DNA onto this substrate
What occurs next and what is the function of southern blotting?
Single stranded DNA probes containing attached radioactive labels of 32P then hybridise with the complementary DNA on the nitrocellulose.
Sheet washed to remove unhybridised DNA.
Photographic film placed onto the nitrocellulose to shoe bands of DNA where the probe has attached,
What is northern blotting?
Same as southern blotting however finding specific RNA sequences
What is western blotting?
Same as southern and northern blotting instead proteins used
What is FISH?
Molecular cytogenetic technique that uses fluorescent probes that bind to specific complementary sequences on the DNA.
FISH function
Localises specific DNA sequences on chromosomes, so can be used in genetic counselling, personalising medicine and species identification.
What can be used to correct the mutated/non functional sequence?
CRISPR - clustered regulatory interspaced short palindromic repeats
What is CRISPR-Cas9?
Consists of a repetition of short sequences of DNA separated by spacer sequences that, along with the Cas9 endonuclease, correspond to an adaptive immune system that protects the bacteria from the invasion of virus or plasmids,
CRISPR-Cas9 DNA technology use
Can be used to add bases or disrupt DNA sequences to alter specific genes with greater accuracy, simplicity and lower cost
Two ways in which Cas9 nuclease breaks the double strand of DNA?
Homology Directed Repair- gene knock in
and Non-Homologous End Joining- gene knock out
Explain Homology Directed Repair
- RNA sequence in CRISPR complementary to the DNA. Cas 9 utilises the sequence to bond with the base pairs on the host DNA.
- Cas 9 causes a double stranded break.
- DNA template then invades the gap , using the DNA’s synthesis machinery to generate a complementary code.
- A segment of DNA from the template strand may be inserted into original break.
- Template separates leaving the original break with a new section of code.
Explain non-homologous end joining
- DNA cleaved by Cas 9
- Either new matching bases added or non-matching bases excised
- joins the ends using ligase
- gene no longer functions
3 enzyme based techniques
Polymerase chain reaction, reverse transcription polymerase chain reaction and DNA sequencing
PCR function
Unlimited amplification of a DNA fragment of interest from a very small amount of a starting template material
PCR stages
- DNA to be amplified sequenced from oligonucleotide fragments
- Added to a thermocycler where temperature is 95 degrees, causing the DNA chains to separate
- Temperature lowered to 50 degrees, enabling the annealing of primers.
- Temperature raised to 72 degrees, enabling taq polymerase to catalyse the condensation of adjacent nucleotides. DNA ligase then joins the Okazaki fragments.
- Cycle is repeated
RT-PCR stages
- Reverse transcriptase enzyme catalyses the formation of a DNA strand from an RNA template.
- A TTTTTTTTTT oligonucleotide primer can be used to transcribe the entire mRNA into DNA, called cDNA
- amplify cDNA using PCR
Function of RT-PCR
Detects and estimates the amount and quality of mRNA
Process where nucleotide sequence of a particular DNA fragment is determined
DNA sequencing
Who discovered the principle of DNA sequencing? + what did he do?
Sanger- determined the nucleotide sequence of insulin
Sanger DNA sequencing requirements
- Requires single stranded DNA template, DNA primer, DNA polymerase, normal deoxynucleotidetriphosphates dNTPs and modified di-deoxynucleotidetriphosphates, ddNTPs.
Sanger DNA sequencing requirements
- Requires single stranded DNA template, DNA primer, DNA polymerase, normal deoxynucleotidetriphosphates dNTPs and modified di-deoxynucleotidetriphosphates, ddNTPs.
A lot more dNTPs are present than ddNTPs in order to completely transcribe the DNA
ddNTPs are tagged using fluorescent dye, each a different colour
Sanger DNA sequencing explained
Using the templates, the complementary DNA strand is being synthesised, however ddNTPs prevent the chain from being further extended, thus producing different lengths of complementary chain.
DNA then denatured whilst heating to release the different length strands.
Then undergo gel electrophoresis and placed onto a radioactive film.
The relative positions of the terminating ddNTPs determine the sequence of DNA, from lightest (start) to heaviest (end)
Different uses of antibodies in protein detection
Western blotting, Elisa, Imaging and Immunoprecipitation
Western Blotting process
- Proteins denatured
- Proteins within the sample are separated using gel electrophoresis, depending upon their molecular mass, electric charge
- Moved from gel to nitrocellulose membrane through the process of electroblotting.
- Membrane is blocked in non fat dry milk or bovine serum. Protein in the solution bind to the membrane in all spaces where the target proteins have not attached.
- antibody is then added to specific target protein and then incubated, washed to remove buffer and unbound antibodies,
- secondary antibody added, linked to probe
- probe deleted using fluorescent detection or radioactive detection.
Western Blot function
Used to detect specific proteins in a sample
What does ELISA stand for?
Enzyme linked immumosorbent assay
ELISA steps
- Antigen added to plate, given time to adhere
- milk added to bind everywhere where antigen is not
- primary antibody with conjugated enzyme added, binds to antigen
- plate washed
- substrate to enzyme added, creating a colour change
- the higher the concentration of primary antibody present, the stronger the colour change
- colour changed measured by spectrometer
ELISA function
Diagnostic tool in medicine
What is ELISA used to screen?
HIV, HIV antigens added, if HIV antibodies present in serum then imdividcl may have HIV.
How are antibodies used in imaging?
Antibodies for a specific protein fluorescently linked
Antibody recognises receptor and binds, glows, can then be detected
Immunoprecipitation definition
The technique of precipitating a protein antigen out of a solution using an antibody that specifically binds to that particular protein
Explain direct immunoprecipitation
- antibodies immobilised onto a specific solid phase, such as a string of breads, such as microscopic agarose
- The beads are then added to the protein mixture where they bind to their targets and are captured
high throughput defined
automation of experiments such that large scale is feasible
