Introduction to Molecular Techniques Flashcards
Give examples of techniques used to analyse DNA at gene level.
RDP: Restriction enzymes DNA gel electrophoresis PCR SMP: Southern hybridisation Microarray PCR variations
Give examples of techniques used to analyse proteins.
PIE:
Protein electrophoresis
Immunoassays
Enzyme assays
Give an example of analysis of DNA at the nucleotide level.
DNA sequencing
Give examples of analysis of DNA at the chromosomal level.
Karyotyping
FISH
What are restriction enzymes?
Usually endonucleases
How do endonucleases differ from exonucleases?
Endonucleases recognise a sequence in the middle of a DNA strand whereas exonucleases recognise a sequence at the end of a strand.
How do restriction enzymes work?
They recognise and cut a specific DNA sequence called a restriction site. These are usually palindromes of 4-8 bp.
Briefly outline DNA gel electrophoresis.
DNA gel electrophoresis is where DNA fragments are put into a gel on a plate. A power supply is used to generate a charge on the plate. At one end where the DNA fragments are put there’s a negative charge. At the other end there is a positive charge. Staining is required to identify the presence of separated DNA fragments. The DNA fragments will therefore move from one side to the other.
Why is an electrical current needed to create a positive end and a negative end?
Because DNA is negatively charged it will move towards the negative side on the other side.
What do the distance between the DNA fragments depend on?
The size of the fragment and the charge. A more negative and smaller fragment will move closer to the positive side.
Why would you use restriction analysis?
To investigate the size of DNA fragments.
To investigate mutations
To investigate DNA variation
To clone DNA
How does gene cloning work?
The relevant gene is cut off by the use of restriction enzymes.
The gene of interest is then put into a plasmid vector called recombinant DNA molecule.
The recombinant DNA is then put into a suitable host like a bacteria.
The bacteria then divides and clones of the gene are produced.
Why would you clone human genes?
To make useful proteins like insulin.
To find out what genes do
Genetic screening
Explain PCR.
DNA is heated up to 95 degrees celsius. This causes the DNA to denature and the strands to separate. At this temperature Taq polymerase which works like DNA polymerase will work at its optimum. The thermostable DNA polymerase (Taq) puts primers forward and reverse on the DNA strands. The DNA is then cooled down again and DNA polymerase will start to copy the DNA strand resulting in two DNA molecules. This can then be done over and over again.
Why would you use PCR?
To amplify a specific DNA fragment
To investigate single base mutations
To investigate small deletions or insertions
To investigate variations and genetic relationships.
What is protein gel electrophoresis?
Similar to DNA gel electrophoresis instead a protein is used.
A gel is used which allows separation of proteins.
A buffer maintains the charge on the proteins.
A power supply which gives a negative and a positive charge.
Staining is used to identify the presence of the separated proteins.
What is the largest difference between DNA GE and Protein GE?
DNA GE is horisontal.
Protein GE is vertical.
How are the proteins separated in Protein GE?
Based on charge, size and shape.
What is isoelectric focusing?
A pH gradient is introduced in a beaker. High pH at the top and low at the bottom. Proteins migrate until they reach a pH equal to their pI (isoelectric point). There’s no charge at pI so the proteins stop migrating.
What is proteomics? (Protein identification)
A way of identifying proteins: Protein is digested by trypsin. Mass spectrometry is performed Generation of a list of peptide sizes A database is used to identify the protein.
What are polyclonal antibodies?
They are produced by many B lymphocytes
Got multiple different antibodies
It’s specific to 1 antigen
They have multiple epitopes
What are monoclonal antibodies?
Produced by 1 B lymphocyte
1 identical antibody
Specific to 1 antigen
1 epitope
How do enzymes assays work?
The product is measured that an enzyme produces.
A continuous assay is done by spectrophotometry
A discontinuous assay is done by radioactivity or chromatography.
