molecular techniques Flashcards
3 processes which allow analysis of DNA
1) restriction enzymes
2) DNA gel electrophoresis
3) PCR
describe how restriction enzymes can be used to analyse DNA
restriction endonuclease cut certain sites on the DNA (the way they cut the DNA results in an overhang). There are specific sites for each restriction endonuclease and these sequences can be found using online programs (because we know full human genome sequence)
How does DNA electrophoresis work?
DNA is put into an agarose gel. Because it is negatively charged it will move towards the anode (+) meaning they will be spirited upon size basis. After this DNA strands are stained so they fluoresce when UV light is shined.
what is DNA electrophoresis used for
to investigate DNA fragment size (see if DNA has deletion etc)
Look at mutations (sickle cell)
Used for DNA fingerprinting (relationships between people)
Used in DNA cloning
describe the process of cloning DNA
(useful in making proteins such as insulin)
1) gene is isolated using restriction endonuclease or reverse transcriptase
2) gene is interred into plasmid vector
3) the plasmid (with the recombinant DNA) is inserted into DNA in bacteria or other stable host
4) bacteria produces the cloned gene and this is isolated and collected
other uses of DNA cloning
gene therapy
genetic screening
to find out what genes do
Describe the process of PCR
DNA amplification.
1) DNA is heated to 95 degrees and h bonds are broken
2) cool back down to room temp allowing primers to aneal to DNA strand
3) after the primers have annealed this allows DNA polymerase to bind and a double strand to be synthesised.
which type of DNA polymerase is used in par and why
tap polymerase because it is thermostable (works at high temps is from bacteria found in hot springs)
why use PCR?
DNA profiling
investigate bas mutations
investigate mutations
all require other process but also require a large small which PCR can provide
3 ways to analyse proteins
1) protein electrophoresis
2) immunoassays
3) enzyme-assays
describe the process of protein electrophoresis
proteins can be seperated on basis of size shape and charge. Proteins run through gel electrophoresis. Then able to compare the banding of proteins from 2 different cells and can see if there is a higher conc of a specific protein in one cell compared to another. (the darker the stain/band the more protein). This may allow diagnosis of certain diseases
what is isoelectric focusing
proteins are separated on the basis of charge. Put in test tube where there is a gradient of pH’s. An electric field is applied and proteins will migrate until they reach a pH which matches their pI. Stop migrating and stained to identify.
what is SDS PAGE
separation based on size
all proteins are denatured so they are the same shape. This is done using SDS which breaks down proteins to secondary structure. B-ME breaks disulphide bonds 1st.
Then all proteins have something applied which gives them the same charge meaning they all move toward the anode. Now carry out gel electrophoresis
what is 2D PAGE
proteins permeated by isoelectric point
this is used in disease diagnosis because able to see up regulation of certain proteins because shows all the individual proteins
what is an immunoassay
using antibodies to bind to specific sites to identify certain proteins.
describe how immunoassays work and used in western blotting
antibodies bind to specific targets produced by B lymphocytes. Nitrocellulose gel is used. Antibody binds to specific protein wanting to be identified (dyed blue) and then an enzyme will bind to the antibody resulting in a single blot where the specific protein is.
what are radioimmunoassays?
The same principle but using radioactively labelled antibodies
what do enzyme assays do
measure enzyme activity
why are enzyme assays useful
finding out how active an enzyme is can be used to diagnose a disease. May be a marker for this disease.
examples of how enzyme assays are used
amylase in blood is an indicator for pancreatitis and troponin 1 is found in blood after MI
describe how Sanger sequencing works
dideoxynucleotides are incorporated into DNA replication but once bound DNA replication is terminated. These nucleotides each contain a different stain depending on which base they are. These DNA strands are then operated by size using gel electrophoresis and able to oder the sizes and bases to find base sequence
describe how southern hybridisation works
DNA probes with radioactive or Uv markers bind to their complimentary sequences. This is then transferred to a sheet using southern blotting. This allows base sequences to be identified. If a base is not present in a gene due to a mutation the probe will not bind
describe how reverse transcriptase works
1) mRNA is isolated
2) primer binds to poly A tail and reverse transcriptase enzyme replicates the mRNA strand going backwards using DNA nucleotides to form cDNA
3) the RNA strand is degraded and use single strand of cDNA to make complimentary strand
4) This is then replicated using PCR
describe how microarray technology works
Used to compare 2 cells (one affected and one non affected). remove mRNA samples and using reverse transcriptase form 2 different sets of cDNA with different stains. Combine these and allow them to hybridise (bind to their complimentary strands on the microchip. Wash the microchip and use a laser to look at the different stains on different genes. Allows you to see which genes are expressed in the affected and unaffected cell.
