Molecular Diagnosis Flashcards
Cloning
Produces exact copies of pieces of DNA, can be used to make useful proteins. Use same restriction enzyme to cut vector and gene. DNA ligase glues sticky ends. Introduce vector with gene into host. Identify and isolate cells containing DNA a of interest.
DNA Gel Electrophoresis
Separates different sized fragments. DNA is negatively charged and moves towards anode if placed in an electric field. Larger fragments move slower therefore less far. (Gel, buffer, power supply & stain)
Restriction Analysis
Use restriction enzymes to cut specific DNA sequences (mostly palindromes). Used to investigate size of DNA fragments/investigate mutations etc.
PCR
Used to amplify DNA fragments/investigate mutations. Heat to 95c to denature, cool to 55c to anneal to primers then heat to 72c for DNA polymerase (TAQ) to synthesise DNA.
DNA Sequencing
Use 4 tubes, each with 1 of the 4 dNTP’s and DNA polymerase. Depending which base is used, will terminate at different places. Producing different sized fragments all ending in a dNTP. Heat fragments, denature and separate via gel electrophoresis. Work out sequence.
SDS-Page
Separates proteins by molecular weight. Add SDS to denature protein (All proteins negative). Put into polyacrylamide gel, use electricity to pull proteins through. Smallest molecules travel furthest.
Isoelectric Focusing
Separation of proteins via charge. Gel has pH gradient, proteins stop moving when they reach a pH equal to their pI.
2D-Page
Isoelectric focusing and SDS-page. Allowing separation of complex mixtures of proteins. Useful for diagnosing diseased state in different tissues.
Western Blotting
After SDS-Page, transfer to nitrocellulose membrane. Antibodies bind to specific target protein. Wash then add another antibody that is specific to the 1st, that is labelled with fluorescence. This is seen as an immunoblot on the gel.
Enzyme-linked Immunoabsorbent Assay (ELISA)
Measures concentrations of proteins. Take sample, put into wells and the proteins bind to the bottom. Add antibody specific to the antigen, and some will bind to the antigens bound to the bottom of the well. Add enzyme-linked 2nd antibody that will then bind to the 1st. Determine how much of the 2nd antibody bound.
Enzyme Assays
Measures how much enzyme is present. Put a substance into a tube, add enzyme and see how much product is produced over time. Measure product via chromatography.
Southern Blotting
Detects particular DNA sequences. DNA fragments firstly undergo gel electrophoresis. Fragments transferred to nitrocellulose membrane. Membrane exposed to hybridisation probe (labelled fragment). Probe hybridises to complementary sequences.
Reverse Transcriptase PCR
RNA a template is used to make complimentary DNA using primer that binds to poly-A tail. Then PCR to replicate.
Microarray
Can look at 1000’s of genes simultaneously, to investigate chromosomal abnormalities. DNA fragments organised into arrays are hybridised to labelled DNA from 2 sources. E.g. To investigate the expression of thousands of species of mRNA in normal and tumour tissue.
DNA Fingerprinting
Everyone has different length of repeating, non-coding DNA which is inherited from you parents. Therefore children share bands with their parents. Based on southern blotting then PCR. Used in forensics/paternity testing.
