Extraction of DNA
Digest the cell wall with lysozyme (for bacteria) and dissolve the cell membrane with detergent
Purification of DNA
Centrifugation: An ultracentrifuge will spin down large molecules, like DNA, because proteins and nucleic acids are denser than water. The S-value (Svedberg unit) is a measure of sedimentation. There are two factors involved: (1) density causes sedimentation and (2) collision with solvent molecules causes dispersion. Therefore intensify centrifugal field to 100,000 x g using ultracentrifuge. Sedimentation velocity is related to molecular weight (MW) and shape for spherical molecules for example: S is proportional (MW)*2/3.
Phenol: Dissolves proteins and is denser than water, so in a test tube, the water layer containing nucleic acids will be above the phenol layer which has the proteins
Ribonuclease: Degrades RNA into nucleotides
Alcohol: Precipitates out intact DNA/RNA/macromolecules. Add alcohol (in the presence of salt), incubate in the cold, and centrifuge to pellet the DNA.
Cutting DNA
A nuclease cuts nucleic acids (DNA or RNA). A ribonuclease (RNase) cuts RNA and a deoxyribonuclease (DNase) - cuts DNA. An exonuclease snips off end of the chain and an endonuclease cuts in middle of the chain.
Restriction Enzymes (RE) cut DNA. They are endonucleases with specific recognition sequences which are inverted repeats. The recognition site is usually 4, 6, or 8 base pairs long. Restriction enzymes are produced by bacterial cells to destroy any foreign DNA (such as viral DNA) that may invade the cell, so they perform a protective role in the cell. Modification enzymes methylate a cell's own DNA so it is protected from restriction enzymes produced by that cell.
|
If RE has a recognition site of (no. of bases) |
You will find a site on average every (bp) |
| 4 | 256 |
| 6 | 4096 |
| 8 | 65536 |
Type I restriction enzymes cut at a site distant from the recognition sequence. They are suicidal enzymes as they can cut DNA only once.
Type II restriction enzymes cut DNA in the middle of the recognition sequence and produce either blunt ends or sticky ends. The sticky ends of two different DNA fragments produced from the same RE will be able base pair and, ultimately, be ligated together.
DNA ligase will seal a nick in a DNA strand and can therefore be used to join two DNA fragments. T4 DNA ligase is able to join fragments with either blunt or sticky ends but bacterial ligases are only able to join fragments with sticky ends.
Electrophoresis
Most molecules are charged, therefore they move in an electric field. The two factors that determine mobility of a specific molecule during electrophoresis are the charge of the molecule (positive or negative) and the surface area (shape) which governs the friction with the solvent.
DNA has a negative charge that is carried on the phosphate group. Negatively charged molecules of DNA will travel at the same speed in a free solution independent of size. To separate the fragments by size, you need a gel which is a crosslinked polymer. This slows the larger molecules and allows the fragments to be separated by size (molecular weight MW) in that smaller DNA fragments are able to migrate faster (and, therefore, further) through a gel than larger fragments. Specifically, mobility is inversely proportional to the log of the MW.
A marker of DNA fragments of known size must be run on a gel with which to compare the unknown fragment sizes. One widely used marker is a kilobase ladder that contains a set of standard DNA fragments which are 1, 2, 3, 4, 5, etc. kb long.
DNA bands that migrate through a gel during electrophoresis are visualized by a process called staining. In the most common staining technique, the gel is stained with ethidium bromide which causes the DNA to fluoresce orange when exposed to ultraviolet light.
Restriction mapping
A single digest (that is, digestion with a single RE) followed by electrophoresis will give the number of cut sites for that enzyme and the distances between these sites (i.e. size of fragments), but it does not give the relative order of the sites. To determine the order of these sites, one must perform a double digest to actually map the restriction sites.
Figure of pBluescript: a ColE1 based plasmid
Detecting a plasmid insert
There are mainly three ways to detect an insert in a plasmid:
1) Extract the plasmid DNA and digest with restriction enzyme. For instance, if you inserted a 4 kb fragment into a 6 kb plasmid, when you digest the recombinant plasmid with a RE that cuts only once and run it on a gel, you should see a 10 kb fragment.
2) Screen for antibiotic resistance. In some plasmid vectors, the multiple cloning site is in a gene for antibiotic resistance and, if insertion succeeds, then the cells containing the recombinant plasmid will lose the antibiotic resistance whereas the cells containing a non-recombinant plasmid would retain the antibiotic resistance.
3) Blue/white color selection. In this scheme, cells that form a blue colony contain a non-recombinant plasmid (just vector) while cells that form a white colony contain a recombinant plasmid (vector and insert). For the color screening, the lacZ gene product is b-galactosidase which can cleave X-gal into galactose and a compound which turns blue when it comes in contact with oxygen. In many cloning vactors, the multiple cloning site (MCS or polylinker; a stretch of DNA containing recognition sites for several restriction enzymes) lies right in the middle of the lacZ gene. This is possible because you can disrupt N-terminus of the protein product and still get functional b-gal. If the gene of interest is inserted into the MCS however, a defective b-gal or no b-gal is produced. Thus, when you screen on plates containing X-gal, cells that produce a functional b-galactosidase (that is, contain an intact lacZ gene) form blue colonies whereas cells that cannot produce b-galactosidase (that is, contain a disrupted lacZ gene) form colonies that are white.
Actually, the way that blue/white color selection works is through a-complementation in which the portion of the lacZ gene encoding the first 146 amino acids (the a-fragment) are on the plasmid and the remainder of the lacZ gene is found on the chromosome of the host. If the a-fragment of the lacZ gene on the plasmid is intact (that is, you have a non-recombinant plasmid), these two fragments of the lacZ gene (one on the plasmid and the other on the chromosome) complement each other and will produce a functional b-galactosidase enzyme. a-Complementation is a very rare event and, in fact, only works with lacZ and a few other genes.
Shuttle vectors
Sometimes it's desirable to clone into a vector which can survive in more than one type of organism. This type of vector is called a shuttle vector. For example, some shuttle vectors are able to function in E. coli (a bacterium) and yeast (a eukaryote). However, selection for a plasmid-containing cell is inherantly more difficult in eukaryotes than it is in bacteria. Yeast, for example, is unaffected by most antibiotics so you can't use antibiotic resistance to select for the plasmid. Therefore, one way to select for the plasmid in yeast cells is to use a yeast host strain with a biosynthetic defect (e.g. leucine requirement). If you then place the gene for leucine biosynthesis on the shuttle vector and omit leucine from selection medium, only cells that contain the shuttle vector are able to survive.
YAC (Yeast Artificial Chromosome)
Because most eukaryotic genes are much larger than prokaryotic genes, you cannot clone these genes using a plasmid cloning system. That is because plasmids will only accept approx. 10 kb inserts at the longest. Some human genes are a million base pairs (a megabase) or more so the only way to clone these very large genes is to use YACs since YACs will accept huge inserts of DNA.
Questions and comments about MICR 302 to: bender@micro.siu.edu
Comments and questions related to web server: webmaster@science.siu.edu
SIUC / College of Science / Microbiology / courses / MICR302
URL: http://www.micro.siu.edu/micro302/techniques.html
Last updated: 17-Mar-99 / laa
