DNA and RNA Structure
Terms in this set (40)
stores genetic information
decodes genetic information during protein syntheses, and has other functions
Who 'discovered the secret of life'?
Watson and Crick (1953)
What joins the phosphate groups together in the DNA backbone?
5-carbon sugar, nitrogenous base, and one or more phosphates
DNA is negatively charged
The phosphate groups make the nucleotides negatively charges, which makes DNA negatively charged
A nucleotide without the phosphate groups
mixture of all four nucleotides
Base pairing in DNA makes two complementary strands
Hydrogen bonds are weak, however, when you have hundreds or thousands of them in a row, it makes a more stable structure. Nevertheless, you don't want the bonds too be too strong, otherwise it would cost way too much energy to unwind DNA for replication and transcription.
What part of DNA is hydrophilic?
What part of DNA is hydrophobic?
nitrogenous bases - they're tucked in the center of the helix so that they're better protected from the water
Why is it important for the edges of the bases in the grooves to interact with protein?
For replication, transcription, and repair.
11 base pairs per turn
narrower, deeper major groove that forms under conditions of low humidity and high salt concentrations, which does not occur for lengthy periods of time in a cell.
double-stranded RNA has this
Watson-Crick DNA - its turns in a clockwise manner and forms under conditions of high humidity and relatively low salt concentrations - the predominant conditions inside a cell.
12 base pairs per turn
It has repeating sequences of CG on one strand, with the complementary CG sequenced on the other, forming a zig-zag helix that can be stable when bound by methyl groups. These helices play an important role in regulating gene expression (they form part of gene regulatory elements).
Differences between DNA and RNA
RNA uses Uracil, whereas DNA uses Thymine
DNA is double-stranded, and RNA is usually single-stranded but can also be double-stranded
RNA's secondary structure
This allows RNA to adopt a certain shape necessary to carry out various reactions/interactions
What direction are DNA sequences written in?
5' to 3'
Organization of genomes in eukaryotes
They package very long molecule of DNA into very small spaces
Packaging unit of DNA
These are important for packing chromosomes to form nucleosomes. Four core histone proteins form an OCTAMER and the DNA wraps around this structure, which creates a nucleosome.
Structural properties of DNA related to its function: "the secret of life"
It has the capacity to store information as well as it is chemically stable. We have 3 million base pairs, meaning that DNA has the capacity to encode huge complexity.
Reasons to exact DNA
Identifying new genes
Analyze transgenic organisms
Analyze gene regulators
Use in diagnostics (such as pathogen detection)
The general strategy used to extract DNA
Release of cell contents
Extraction (from proteins, lipids, and cell debris)
Purify and concentrate the DNA
1. Mechanical techniques such as grinding is used to homogenize the DNA sample
2. Detergents such as SDS and chaotropic agents, which removes the membranes, are used to chemically lyse the homogenized sample
3. If mammalian cells are use, then they must be out in water, which causes the cell to burst due to osmotic shock. This allows the cell contents to be released
4. Enzymes such as proteinase K are used to digest the proteins
5. The DNA is then separated from proteins, lipids, and other cellular debris through the addition of NaCl.
The result is: cell lysate
Extraction by Spooling
Ethanol is added to the cell lysate to force the DNA to precipitate in the solution and to prevent the DNA from dissolving in water. DNA then collects at the interphase between the lysate and the ethanol since DNA is insoluble in ethanol. The solution is then filtered in order to separate the supernatant from the cell lysate. Once filtered, IMS (alcohol) is added into the supernatant and a spool is then used to isolate the DNA.
Using spectrometry to estimate the concentration of a sample of pure DNA
DNA absorbs UV light at 260nm.
Optical density = 1.0, which means there is 50 micrograms per mL of dsDNA.
SO if a dsDNA extraction has an absorbance of 0.5 at 260, then the concentration of the DNA must be 0.5 x 50 micrograms/mL = 25 micrograms/mL
Estimating the purity of DNA
The ratio between the absorbances at 260 and 280 allows us to determine the purity of the sample of DNA
The ratio of pure DNA is approximately 1.8
If the ratio is less than 1.6 this means that the sample may be contaminated other materials such as proteins, lipids, and phenols.
The melting of DNA
DNA stranded that are more concentrated with nitrogenous bases C and G have a higher melting point than those with nitrogenous bases A and T. This is because bases C and G form three hydrogen bonds between them, whereas A and T only form two hydrogen bonds.
This theory is based upon the principle of the kinetics of DNA renaturation. Renaturation is dependent on the concentration of DNA, the reassociation temperature, and the cation concentration.
What does the annealing of DNA tell us about its structure?
Sequences with common or repetitive sequences renature more rapidly than those with rare or single-copy sequences. This information can tell us about the genome size and relative proportions of single-copy and repetitive sequences.
association of two single strands to form a duplex - nucleic acids from different sources are involved and renature (hybridize)
the ability of two single strands to hybridize is dependent on their complementarity
tools for hybridization
a labelled probe strand can be used to detect an unlabelled target strand
Filter membranes can be used to immobilize target sequences through the use of UV cross-linking or heat, causing the target sequences to covalently link to these membranes
Southern Blotting is used to identify specific dsDNA fragments that have been size-fractioned by agarose gel electrophoresis. DNA that has been size fraction and usually digested by restriction enzymes is separated on a gel. This gel is then treated with an alkali in order to denature the DNA and then the DNA image of the gel is transferred to a membrane by capillary, vacuum, or centrifugal blotting. The DNA probe (radio-labelled DNA) is added and picks out the specific target DNA and hybridizes. The membrane is washed and undergoes autoradiography in order for the bands to be visualized.
This method can be used to identify gene rearrangements in normal versus diseased states
This process is very similar to the Southern Blotting, however, the major difference is that RNA is analyzed, rather than DNA. Nevertheless, the probe used in this can either be DNA or RNA.
Since RNA is used, this process can be used to determine gene expression in different tissues or expression levels in normal versus a diseased state
Fluorescence In Situ Hybridization (FISH)
This method is used to detect and localize the presence and absence of specific DNA sequences on chromosomes. In this process, cells are immobilized on a slide and the signals are detected by fluorescence or confocal microscopy.
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