Ch. 5 - Manipulation of Nucleic Acids Flashcards
Nucleases
Enzymes that cut or degrade nucleic acids.
Deoxyribonucleases (DNase): Nuclease that attacks DNA.
Ribonuclease (RNase): Nuclease that attacks RNA.
Exonucleases: Attack/cleave at the end of the nucleic acid molecules, and usually remove just a single nucleotide, or sometimes a short piece of single-stranded DNA. Exonucleases can attack either the 3´-end of the 5´-end, but not both.
Endonucleases: Attack/cleave nucleic acid molecules in the middle. Some endonucleases are non-specific; others, in particular the restriction enzymes, are extremely specific and will only cut DNA after binding to specific recognition sequences.
Restriction enzymes
Endonuclease that cuts double-stranded DNA at a specific sequence of bases, the recognition site. Most recognition sites for restriction enzymes are inverted repeats of 4, 6, or 8 bases. Restriction enzymes recognize sequences of 4-8 nt in length on foreign DNA and cut both phosphate backbones to break the single piece into two. Restriction enzymes can be divided into two major classes based on where they cut the DNA, relative to the recognition site: Type I and Type II restriction enzymes.
Bacteria produce restriction enzymes to destroy foreign DNA, and restriction enzymes have names derived from the initials of the bacteria they come from (First letter of genus, capital, followed by two first letters of species).
Isoschizomers: Restriction enzymes from different species that share the same recognition sequence. These may not always cut in the same place even though they bind to the same base sequence.
Modification enzymes
Restriction enzymes also need to recognize bacterial DNA to successfully degrade the foreign DNA without endangering the bacterias own DNA. To protect their own DNA, bacteria have modification enzymes that can distinguish between their own DNA and the foreign DNA.
Modification enzymes recognize and bind to the same recognition site, and transfer a methyl group onto adenine or cytosine residues within the recognition site on the DNA. The methyl group prevents the corresponding restriction enzyme from recognizing the site. Consequently, the bacterial DNA is immune to the cell´s own restriction enzymes, while incoming, unmodified DNA will be degraded by the restriction enzyme.
Type I restriction enzyme
Cuts the DNA 1000 or more base pairs away from the recognition site. This is done by looping the DNA around so that the enzyme binds both at the recognition site and the cutting site. Type I restriction systems consist of a single protein with three different subunits. One subunit (specificity) recognizes the DNA, another (modification) methylates the recognition sequence, and the third (restriction) cuts the DNA at a distance from the recognition site. Due to inconsistency of the length of the loop, and as the base sequence at the cut site is not fixed, Type I restriction enzymes are of little practical use. In addition, Type I restriction enzymes are suicidal, and can only cut DNA once before its inactivated.
Type II restriction enzymes and Sticky ends
Cuts the DNA within the recognition sequence. There are two different ways of cutting the recognition site in half. One way is to cut both strands of the DNA at the same point, leaving blunt ends. Blunt ends are fully base paired, and have no unpaired single-stranded overhang. The alternative is to cut the two strands in different places, which generates unpaired single-stranded overhanging ends. The ends made by such staggered cut are free to base pair with any complementary sequence, and consequently are known as sticky ends.
In Type II restriction systems, the restriction endonuclease only cuts DNA, unlike Type I restriction enzymes that have multiple enzymatic functions. Since the exact position of the cut is known, these restriction enzymes are normally used in genetic engineering.
Enzymes that generate sticky ends are the most useful. If two different pieces of DNA are cut with the same restriction enzyme, or even with different enzymes that generate the same overhang, the same sticky ends are generated. This allows fragments of DNA from two different original DNA molecules to be bound together (temporary) by matching the sticky ends. These fragments can be ligated (permanently) together by DNA ligase. Sticky ends are more convenient than blunt ends when joining fragments of DNA using DNA ligase. When two sticky ends made by the same enzyme are ligated, the junction may be cut apart later by using the same enzyme again. However, if two sticky ends made by two different enzymes are ligated together, a hybrid site is formed that cannot be cut by either enzyme.
DNA ligase
Enzyme that joins DNA fragments covalently, end to end. If DNA ligase finds to DNA fragments touching each other end to end, it will ligate them together. The binding between two sticky ends is temporary, as it consists of only hydrogen bonds. To keep the pieces permanently together, DNA ligase is used to ligate the sticky ends together through covalent phosphodiester bones in the backbone.
Ligating blunt ends is very slow and requires high concentrations of both DNA and DNA ligase. Bacterial ligase cannot join blunt ends at all. T4 (viral) ligase is normally used in genetic engineering as it can join blunt ends if need be.
Restriction map
A diagram showing the location of cut sites on a segment of DNA for a variety of restriction enzymes. To generate such a map, DNA is digested with a series of restriction enzymes, one at a time. The fragments of digested DNA are separated by (agarose) gel electrophoresis. Comparison with appropriate standards allows the sizes of the fragments to be estimated.
To determine the order/organization of the fragments and thereby the cut sites, double digests using two restriction enzymes are performed. Restriction maps are deduced by cutting the target DNA with a selection of restriction enzymes, both alone and in pairs.
Gene cassette
Deliberately designed segment of DNA that is flanked by convenient restriction sites and carries a gene for resistance to an antibiotic or some other easily detected/observed characteristic.
A gene to be disrupted is cut with a restriction enzyme. An artificially constructed cassette that confers antibiotic resistance is inserted into the cut site and ligated into the gene. The new DNA construct formed can be detected easily since it provides resistance to antibiotics.
Restriction Fragment Length Polymorphism (RFLP)
A difference in the sequence at a restriction enzyme recognition site between two related DNA molecules that results in production of restriction fragments of different lengths after a restriction enzyme digest. RFLPs may be used to identify organisms or analyze relationships, even though we do not know the function of the altered gene, and are widely used in forensic analysis.
The recognition sequence for a particular restriction enzyme is extremely specific. Changing a single base can prevent recognition and cutting.
Synthesis of whole genes
Whole genes have been made by synthesizing smaller, overlapping fragments (oligonucleotides) and then assembling them.
If the gaps between the fragments are small, they can be assembled by annealing them, and then seal the nicks with DNA ligase.
If the gaps are bigger, they can be assembled by annealing them and then filling the gaps with DNA polymerase I, and finally seal the nicks with DNA ligase.
When designing a gene, the gene has to be cloned into a plasmid and sequence it to validate that it is the right one.
It gets easier to make mistakes as the gene gets longer.
High throughput, fragments can be bought by companies.
Quantification of DNA and RNA by UV light
The concentration of DNA or RNA can be measured by absorption of ultraviolet (UV) light, as the aromatic rings in the bases of DNA and RNA absorb UV light with an absorption maximum at 260 nm. The phosphate backbone is not involved in UV absorption. The concentration can be measured by using spectrophotometry by comparison with a standard curve.
Proteins absorb UV light at 280 nm. The relative purity of a preparation of DNA can be assessed by measuring its absorbance at both 260 and 280 nm and computing the ratio.
Pure DNA has an A260/A280 ratio of 1,8.
If protein is present, the ratio will be less than 1,8.
If RNA is present, the ratio will be greater than 1,8.
Pure RNA has an A269/A280 ration of approx. 2,0.
The structure of the nucleic acid dictates how much light the aromatic rings absorb.
When the nucleotides are free, they can spread out such that each ring can absorb the UV light. Overall, the free nucleotides absorb more UV light.
When the nucleotides are bound to each other in a nucleic acid polymer, and the aromatic rings are stacked along the phosphate backbone, the rings shield each other and absorb less UV light.
Pulse-field gel electrophoresis
Used for separation of large DNA molecules, molecules too large for the pores in agarose gel. Electric pulses switch between two diagonal fields, and large molecules can then “wiggle” through the matrix.
Radioactive labeling of DNA and RNA
Radioactive isotopes are used to label DNA or RNA. The most common isotopes used are those of sulfur (35S) or phosphorus (32P). The radioactive phosphorus is inserted into the nucleic acid as a substitute to the phosphate group. The radioactive sulfur is inserted bound to the phosphate group (phosphorothioate).
“Hot”: radioactive
“Cold”: non-radioactive
This method is not used a lot anymore as there are better ways.
Scintillation counting
Method to detect and measure radioactivity. Detection and counting of individual microscopic pulses of light. Used if the sample is in liquid form, or on a strip of filter paper. The method relies on special chemicals called scintillants that are mixed with the nucleic acid sample. Scintillants are molecules that emit pulses of light when hit by high energy electrons, known as beta-particles, that are released by the radioactive isotopes in DNA. The light pulses from the scintillant are detected by a photocell, and registered by a scintillation counter. Scintillation counting is a Quick and good method for detection of radio-labeled DNA.
Autoradiography
Method to detect and measure radioactivity. Used to locate radioactive molecules on gels or membranes. If the sample is flat, such as an agarose gel, autoradiography can be used to pinpoint the location of radioactive bands or spots.
Can be used for detecting radioactively-labeled DNA or RNA in a gel after separation by electrophoresis. The gel is dried and a piece of photographic film is placed on top of it. The two are loaded into a cassette case that prevents light from entering. After some time (hours to days), the film is developed, and dark lines appear where the radioactive nucleic acids were present, revealing the position of the bands. This is because beta-particles released from the radioactive isotopes stain the photographic film. Used to visualize DNA fragments on a gel after electrophoresis.
