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BIOL 308
Dragana Miskovic

1. What kind of information can we obtain from a northern blot? northern blotting is used to detect RNA sequences. It can be used to detect a specific mRNA fragment from a specific organ at a specific time. By changing the conditions and the controlling factors (spatial and temporal), the effect of these factors on mRNA can be analyzed. 2. What kind of information can we obtain from a Southern blot? Southern blotting is used to detect DNA sequences. It can be used to: a) Detect specific DNA fragments b) Detect the number and position of gene copies in the genome c) Detect restriction mapping of fragments d) Detection of cloned sequences e) Detection of transgenes f) Detection of homologous sequences in different genomes g) Detection of repetitive sequences h) Used for genotyping 3. Compare and contrast Southern and northern blotting technique. Northern Southern RNA is already single stranded DNA has to be denatured Detects steady state level of a specific transcript Genomic DNA levels are the same at all times. a certain RNA mixture. (Spatial, temporal and conditions change the level of mRNA) Depends on both transcription and degradation rate DNA is stable and is not degraded under normal for that specific mRNA physiological conditions. Different buffer from Southern Different buffer from northern. N.B: In situ hybridization is used to identify genes directly in chromosomes and transcripts (mRNA) directly in chromosomes. 4. Compare the information obtained by northern analysis with the information obtained by microarray experiments. Northern analysis provides information about one particular transcript under the condition tested. However, microarrays provide information of the total gene expression in a cell and the interaction of the gene products. They can also be used to find out which genes are expressed and which aren’t. N.B: In microarrays, the probe is known, its fixed and unlabeled, whereas the target are the unknown sequences that are labelled during the experiment. They are mobile. (cDNA made from mRNA) 5. How do we treat a DNA gel prior to Southern blotting? Explain why. 6. Thinking question: you have made a short probe (50 nucleotides) from a certain genomic DNA (note: genomic DNAs include both exons and introns). Are you sure that you would be able to use this probe for northern hybridization? Explain your reasoning. The DNA is first collected then isolated from other proteins, cellular debris and RNA. Restriction enzymes are used to cut the DNA into fragments (otherwise, DNA fragment is too heavy and will not move on the gel) the DNA is then denatured using heat or alkaline conditions. This is done because dsDNA will not hybridize to the probes. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. Current is allowed to pass. The negatively charged DNA fragments move towards the anode with a speed inversely proportional to their size. A sheet of nitrocellulose membrane is placed on top of the gel. Pressure is applied evenly to the gel by placing a stack of paper towels and a weight on top of the membrane and gel, to ensure good and even contact between gel and membrane. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane. The membrane is then baked, i.e., exp
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