MCBG Session 12 - Protein Function and Targeting Flashcards
Discuss the importance of restriction endonucleases.
- One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecules involved.
- The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded (ds) DNA into smaller, more manageable fragments, has opened the way for DNA analysis.
- Because each enzyme cleaves DNA at a specific nucleotide sequence, restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments.
What are “sticky” ends and what do they do?
- Restriction enzymes cleave dsDNA so as to produce a 3’-hydroxyl group on one end and a 5’-phosphate group on the other.
- Some restriction endonucleases, such as TaqI, form staggered cuts that produce “sticky” or cohesive ends—that is, the resulting DNA fragments have single-stranded (ss) sequences that are complementary to each other.
- Using the enzyme DNA ligase, sticky ends of a DNA fragment of interest can be covalently joined with other DNA fragments that have sticky ends produced by cleavage with the same restriction endonuclease.
What are restriction sites?
- A DNA sequence that is recognized and cut by a restriction enzyme is called a restriction site.
- Restriction endonucleases cleave dsDNA into fragments of different sizes.
- For example, an enzyme that recognizes a specific four-base-pair sequence produces many cuts in the DNA molecule, one every 44 bp.
- In contrast, an enzyme requiring a unique sequence of six base pairs produces fewer cuts (one every 46 bp) and, hence, longer pieces.
- Hundreds of these enzymes, having different cleavage specificities (varying in both nucleotide sequences and length of recognition sites), are commercially available as analytic reagents.
What is DNA cloning? Outline the steps involved.
- Introduction of a foreign DNA molecule into a replicating cell permits the cloning or amplification (that is, the production of many identical copies) of that DNA.
- By this mechanism, many identical copies of the DNA of interest can be produced. Hence, summary of steps:
I. Isolate relevant gene of interest following digestion with restriction enzymes
II. Insert gene of interest into plasmid vector (recombinant DNA molecule)
III. Introduce recombinant DNA molecule into suitable host cells e.g. E. coli
IV. Identify and isolate the clone containing the DNA of interest
What is the purpose of DNA cloning?
- To make useful proteins e.g. insulin
- To find out what genes do e.g. HTT
- Genetic screening e.g. Huntington’s, BRCA1/2, Cystic Fibrosis
- -* Gene therapy ? e.g. Cystic Fibrosis
What is a vector?
- A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined.
- Essential properties of a vector include:
1. It must be capable of autonomous replication within a host cell;
2. It must contain at least one specific nucleotide sequence recognized by a restriction endonuclease;
3. It must carry at least one gene that confers the ability to select for the vector, such as an antibiotic resistance gene. Commonly used vectors include plasmids and viruses.
What are prokaryotic plasmids?
- Prokaryotic organisms typically contain single, large, circular chromosomes.
- In addition, most species of bacteria also normally contain small, circular, extrachromosomal DNA molecules called plasmids.
- Plasmids may carry genes that convey antibiotic resistance to the host bacterium, and may facilitate the transfer of genetic information from one bacterium to another.
- Plasmids can be readily isolated from bacterial cells, their circular DNA cleaved at specific sites by restriction endonucleases.
- The recombinant plasmid can be introduced into a bacterium, and large numbers of copies of the plasmid produced.
- The bacteria are grown in the presence of antibiotics, thus selecting for cells containing the hybrid plasmids, which provide antibiotic resistance.
What is PCR?
- The polymerase chain reaction (PCR) is a test tube method for amplifying a selected DNA sequence that does not rely on the biologic cloning method.
- PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours.
- It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample.
- The method can be used to amplify DNA sequences from any source—bacterial, viral, plant, or animal.
- It uses thermostable DNA Polymerase (Taq DNA Polymerase).
- It involves a pair of primers (forward and reverse), uniquely defining the region to be copied.
- Temperature cycles of denature, anneal, and polymerise.
- Repeated copying results in exponential increase in DNA.
What is the purpose of PCR?
- To amplify a specific DNA fragment
- To investigate single base mutations e.g. Tay Sachs, Sickle Cell disease
- To investigate small deletions or insertions e.g. Cystic Fibrosis
- To investigate variation, genetic relationships e.g. DNA profiling, DNA typing
Outline the steps involved in Northern blots.
- Northern blots are very similar to Southern blots, except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized to a radiolabelled probe.
- The bands obtained by autoradiography give a measure of the amount and size of particular mRNA molecules in the sample.
Outline the steps involved in Microarray analysis of gene expression.
- DNA microarrays contain thousands of immobilized DNA sequences organized in an area no larger than a microscope slide.
- These microarrays are used to analyse a sample for the presence of gene variations or mutations (genotyping), or to determine the patterns of mRNA production (gene expression analysis), analyzing thousands of genes at the same time.
- For genotyping analysis, the cellular sample is genomic DNA. For expression analysis, the population of mRNA molecules from a particular cell type is converted to cDNA and labelled with a fluorescent tag.
- This mixture is then exposed to a gene chip, which is a glass slide or membrane containing thousands of tiny spots of DNA, each corresponding to a different gene.
- The amount of fluorescence bound to each spot is a measure of the amount of that particular mRNA in the sample.
- DNA microarrays are often used to determine the differing patterns of gene expression in two different types of cell—for example, normal and cancer cells.
- Physicians hope to one day be able to tailor treatment regimens to each cancer patient, based on the specific microarray expression patterns exhibited by that patient’s individual tumour.
Outline the steps involved in the process of Enzyme-linked immunosorbent assays (ELISA).
- These assays are performed in the wells of a plastic microtiter dish.
- The antigen (protein) is bound to the plastic of the dish.
- The probe used consists of an antibody specific for the particular protein to be measured.
- The antibody is covalently bound to an enzyme, which will produce a coloured product when exposed to its substrate.
- The amount of colour produced can be used to determine the amount of protein (or antibody) in the sample to be tested.
- They can be used to measure the concentration of a protein in solution e.g. hormones
Outline the steps involved in Western blots.
- Western blots (also called immunoblots) are similar to Southern blots, except that protein molecules in the sample are separated by electrophoresis and blotted (transferred) to a membrane.
- The probe is a labelled antibody, which produces a band at the location of its antigen.
Outline the steps involved in proteomics.
- Proteomics: The study of all proteins expressed by a genome, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, is known as proteomics.
- The 20,000 to 30,000 protein coding genes of the human genome translate into over 100,000 proteins when posttranscriptional and posttranslational modifications are considered.
- Although a genome remains unchanged, the amounts and types of proteins in any particular cell change dramatically as genes are turned on and off.
- Proteomics offers the potential of identifying new disease markers and drug targets.
Compare and contrast proteomics with molecular diagnosis
- Proteomics: analysis of all proteins expressed from genome
- Molecular diagnosis: analysis of a single purified protein
Compare and contrast polyclonal and monoclonal antibodies.
- Polyclonal antibodies:
I. Produced by many B lymphocytes
II. Multiple different antibodies
III. Specific to 1 antigen
IV. Multiple epitopes
- Monoclonal antibodies:
I. Produced from 1 B lymphocyte
II. 1 identical antibody
III. Specific to 1 antigen
IV. 1 epitope
Outline the components and process of DNA gel electrophoresis.
- DNA is negatively charged and will move towards the anode if placed in an electric field
- DNA fragments can be separated on the basis of size (or shape)
- Requirements for gel electrophoresis:
I. Gel - A matrix that allows separation of DNA fragments
II. Buffer - Allows charge on the DNA samples across the gel
III. Power supply - Generates charge difference across the gel
IV. Stain/detection - To identify the presence of the separated DNA
Outline the components and process of protein gel electrophoresis.
- Proteins are charged molecules and will move towards the anode or the cathode if placed in an electric field.
- Proteins can be separated on the basis of size, shape or charge
- Requirements for gel electrophoresis:
I. Gel - a matrix that allows separation of the protein sample
II. Buffer - maintains charge on the protein samples
III. Power supply - generates charge difference across the gel
IV. Stain/detection - to identify the presence of the separated proteins
What is Isoelectric focusing (IEF)?
- Proteins separate on the basis of charge
- Proteins migrate until they reah a pH equal to their pI
- No net charge at pI so stop migrating
What is Two dimensional electrophoresis (2D-Page)?
- Allows the separation of comple mixtures of proteins
- Important for diagnosisng disease states in different tissues
Outline serum protein electrophoresis.
What is Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS Page)?
Provide examples of continuous and discontinuous assays.
- Continuous assays:
I. Spectrophotometry
II. Chemoluminescence
- Discontinuous assays
I. Radioactivity
II. Chromatography
