In this unit, we will discuss the following technologies:

1. Cloning DNA sequences into plasmids
2. Use of expression vectors
3. Cloning genomic fragments
4. Cloning RNA molecules using reverse transcriptase
5. Dot blot analysis
6. Simple restriction mapping
7. Southern blot analysis
8. Northern blot analysis
9. DNA fingerprinting
10. Case studies using DNA fingerprinting
11. Chromosome walking
12. PCR analysis
13. DNA sequencing

What good is having a plasmid?

• The DNA of a human, or even a Drosophila, is too complex to study, directly.
• A plamid is a small and well defined DNA molecule.
• DNA can be inserted into a plasmids; the plasmids can be replicated within bacteria; and, then the plasmids can be retrieved from the bacteria. Large quantity of plasmid DNA can be generated in just a few hours.
• Plasmids can be used to express the DNA that has been inserted.
• Plasmids can be used to store DNA for later use, and in a small-size package.
• Plasmids make it easy to purify the DNA that was inserted.

How do you insert DNA into a plasmid?

• Restriction enzymes, which are actually site-specific endonucleases, can cut DNA at a specific recognition site. Each restriction enzyme is able to identify and cut at a certain site. See Figure 19.1.
  • Usually, restriction enzymes recognize palindromic sequences of 4 to 6 bases in length.
  • A few (3 so far, the last time I looked) can recognize 8 base pairs.
  • An example of a palindromic sequence recognizable by a restriction enzyme would be the sequence 5'CCCGGG3'.
  • Write the complementary sequence and read it from 5' to 3', see that it reads the same way.
  • A certain restriction enzyme would recognize that sequence. In fact, for any given site more than one enzyme might exist. But the enzymes that do cut this site would not cut other sites, except a site like 5'ACCCGGGT3' for example, because it contains 5'CCCGGG3'.
• One of the types of enzymes that recognize the site might cut the DNA in the middle leaving two pieces of DNA that are both double. A different type might cut symmetrically away from the middle, resulting in two double-stranded DNA molecules, with short complementary single-stranded ends.
• Restriction enzymes are isolated from bacteria. Different strains and species of bacteria have been found to possess them. The enzyme's "catalog" name, by convention, includes information about the species from which it was isolated. The restriction enzyme EcoR1 for example was isolated from E. coli.
• Plasmids are well defined, at least the ones you order from companies.
• All known restriction enzymes have been used on each certain plasmid, and a map of restriction sites is provided in the catalog.
• We will discuss how to restriction map later.
• If you cut a plasmid with a restriction enzyme that leaves staggered, single-stranded ends, and cut the DNA that needs to be cloned with the same enzyme, when the two are mixed, the DNA to insert will sometimes complementary hydrogen bond to the plasmid DNA. Having the right concentration of the two helps; and, there are "cookbooks" that will give you the right concentrations.
• The DNA to insert and the plasmid DNA are added together in a tube. Later, ligase is added to seal the molecules together.
• You can guess that the next step involves the transformation of bacteria with this manipulated DNA. The bacteria are grown for a little while, and then selected against using an antibiotic the plasmid confers resistance to.

How do you know that the DNA you wanted inserted actually got into the plasmid?

• There are many ways, generally they involve screening.
• With selection, only one of a million might be what you want for something. You set up the system so that only the desired ones grow. Individuals don't need to be inspected.
• With screening, each clone from a transformation is evaluated.
• If the plasmid had two antibiotic genes, one would be used to select for bacteria that took up the plasmid, whilst the other might be used to determine if the DNA is in fact in the plasmid.
• First, select with the antibiotic that the plasmid would confer resistance to, then screen each of the colonies (clones) to see which ones have lost the other antibiotic resistance.
• The restriction enzyme would have cut within the screenable marker and if the insert did enter that region, the event would have inactivated the gene in that region.
• For example, the streptomycin gene was the intact and is the selectable marker. Another antibiotic resistance gene might be for kanamycin.
• Take colonies from the plate used to select and transfer them point for point to a plate containing kanamycin. If the colonies fail to grow on the kanamycin plate, they contain the insert and are the ones you want.
• Take the missing colonies from the strep plate and grow them.
• If you know the size of the inserted DNA you can further verify that the insert is there using gel electrophoresis.
  • Grow the purported genetically engineered bacteria, isolate the plasmids, and cut them with the same enzyme used to insert the DNA.
  • Run the DNA on an electrophoreses gel, which separates molecules based on molecular weight. The DNA runs toward the positive pole. (Why?)
  • Check the molecular weights of DNA fragments by comparing to known markers run along side of the plasmid DNA.
  • If two bands appear in the gel, great. If the bands are the correct molecular weight, greater.
  • How do you see the DNA in the gel? Stain the gel with ethydium bromide and place it on a UV light box (called a transilluminator).
• This is how you "clone" DNA in basic terms.
• An additional note about restriction enzymes:
  • A long DNA molecule of random base pair composition will be cut into smaller fragments by a restriction enzyme. The average size of the fragment depends on how many bases the restriction enzyme recognizes and the amount of each of the recognized bases. For example, let's assume that there is an equal amount of the bases C and A ( and so all bases are in equal concentration). The sequence AGCT would occur in the DNA, and be cut by the enzyme that recognizes it, once every 256 bases (A:1/4;G:1/4;C:1/4;T:1/4, and therefor, 1/4X1/4X1/4X1/4=1/256). What would have been the results if the base A was found 15% of the time?
  • Therefor, the average size of the fragments generated by a restriction enzyme is due to the number of bases recognized and chance.

• Briefly, the plasmid in this case would include a promoter region around the DNA insertion restriction site.
• Cut, insert and ligate as before.
• Often, plasmids will include a screenable marker on the other side of the promoter. The promoter is out of frame until you insert your fragment. When you clone your fragment into the plasmid, you make a hybrid gene. The first or last part is your DNA and the other codes for the screenable marker. The protein is hybrid also.
• The gene expression can then often be regulated. See Figure 19.16.

• This is where the genius to molecular biology comes into play.
• There are as many strategies to isolating certain DNA fragments as there are fragments that people want.
• But, if any human DNA fragment will do, just cut the isolated DNA from a person with the same enzyme used to cut the plasmid at the insertion site, and lygate them together. Once you have the DNA fragment cloned, you can save it forever. See Figure 19.9. Or, maybe clone DNA from the blood cells found in an amber-encased mossy.
• This DNA would be called genomic DNA because it comes straight from a genome.

• Human genes can be cloned using mRNA and reverse transcriptase.
• We have covered both of these before. Reverse transcriptase is a gene found in retroviruses. It synthesizes DNA from RNA templates.
• A primer is needed again, just as for all DNA polymerases. A synthetic primer of poly T is used to complement the 3' poly A tail of eukaryotic DNA. It's almost too good to be true! See Figure for actual details, which includes the use of DNA poly I and S1 nuclease.
• The resulting DNA is called cDNA for complementary DNA. Another great thing about cDNA is that it is minus the introns. Bacteria cannot remove introns. The product produced from the cDNA in the bacteria is amino acid base perfect for what is produced in the organisms, except for any post translation modifications that might be required by some proteins.

This is a technique that provides a means to determine if a sequence is present in a cell, or which cells have the sequence.

• Making Probe:
  • The sequence of interest has been cloned previously and is now in a plasmid.
  • Grow the bacteria that have the plasmid.
  • Isolate the plasmids.
  • Nick translate the plasmid.

• Introduce nicks into the plasmid DNA. Nicks are breaks in the phosphodiester linkage. If the number of nicks are kept low enough, none or very few will be across from each other so the plasmid will stay in one piece due to hydrogen bonding between complementary strands.
• Add radioactive labeled nucleoside triphosphates (_³²P).
• Add DNA polymerase I; you know what will happen next from our previous lessons.
• Add ligase.
• This is now a probe for the unique sequence of interest.
• Preparing DNA Blots

• Take cells in question and isolate their DNA.
• Blot the DNA to a special piece of paper or nylon.
• After treating the paper so that the DNA attaches, treat it so no more DNA attaches to the paper.

• Hybridize the Blot DNA with the Probe DNA
  • Put the paper with the DNA attached into a plastic bag; add hyb solution, and radioactive-labeled probe; put everything into a hybe oven.
  • Heat and cool cycle to hybridize the probe DNA to the DNA that is stuck to the paper.
  • Open the bag; wash the paper to remove all unbound probe; dry paper.
  • The primer is longer than the homologous region of the DNA that is attached to the paper. The extra just hangs around adding to the strength of the signal.
• Finding where the radioactivity is on the paper.
  • Place it into a standard X-ray film canister that is loaded with film.
  • Let set for 24 hours.
  • Develop the film. Examine for spots.
• Interpreting your results
  • Wherever spots appear, hybridization occurred on the paper.
  • Where hybridization occurs, the sequence of interest is present.

An example of the use of dot blots would be the example given in class about whether or not a certain sequence is Y-chromosome specific. See Figure 19.12.

• We learned how to map genes in Drosophila, bacteria, and even phage.
• Mapping restriction sites in DNA fragments is an even finer level of mapping.
• It is extremely useful, and its uses have been extended to include assignment of biological samples to a certain individual, proving parentage, determining the likelihood of contracting a disease, evaluating genetic diversity in plant and animal populations, and more.
• Restriction mapping is also an important step in the DNA sequencing process. Long pieces of DNA cannot be sequenced directly. They must be broken up into smaller pieces, the smaller fragments can then be sequenced and with a restriction map, the smaller pieces can be reconstructed to provide the complete sequence for a region of DNA.
• In terms of getting the actual DNA sequence for a gene, for example, restriction mapping is one level above the actual sequencing level, which is of course the finest level.
  • There are two main ways to conduct restriction mapping. Incomplete digests and digestions that involve more than one restriction enzyme, called double or triple digest depending on whether 2 or 3 enzymes are used in the analysis.
    • The incomplete digest method involves using one restriction enzyme on the DNA sample. One treatment involves letting the digestion go to completion. Another treatment stops the digestion before completion.
    • The smaller fragments found in the complete digestion must be part of the larger fragments found in the incomplete digestion. For example if the restriction site that generates 10 kb fragments is next to the restriction site that generates 15 kb, some 25 kb fragments would be found in the incomplete digest. From the presence of the 25kb fragments, and the 15 and 10kb fragments in the complete digest, you could determine the order of restriction sites and the distances between.
  • The other method, double or triple digest, involves the same basic logic.
    • Cut the DNA with each of the enzymes separately in one treatment, and cut with all the enzymes in another treatment.
    • The smaller pieces from the combination treatment must come from the larger fragments of the single.
    • Analyzing these results allows you to organize the restriction sites for the different enzymes used for the DNA that was cut. See Figure 19.26.

Simple restriction analysis, for example, of plasmid DNA, can be assessed for length using standard gel elctrophoresis, DNA staining and viewing using a transilluminator. See Figure 19.19.

Southern blots are a combination of the procedures described for the dot blots and the ones from the simple restriction mapping above.

The problem is that if you cut DNA from a human with a restriction enzyme that recognizes 6 bp, too many fragments would be generated to visualize on a gel. How many? More than 3,000,000,000/(4X4X4X4X4X4). More fragments because the 3 billion is for the haploid genome, and the other copy would cut somewhat differently, adding to the total number of fragments.

The average fragment length would be the denominator from the above equation. However, due to random (and nonrandom) chance, there would be a range of sizes.

If you ran this DNA on a gel and stained it, it would simply create a smear.

So what is done is to make a probe, like with the dot blot procedure.

• DNA is cut with one or two restriction enzymes and run on a gel.
• The DNA from the gel is then transferred point for point to the blotter paper.
• The paper is then "probed" as it was in the dot blot analysis.
• The same x-ray film technique is used to visualize the location of the radioactive molecules.

Bands will appear where the probe stuck. Analysis of various sorts can be done on the bases of these. See Figure 19.20.

Northern Blots are just like southern, but involve RNA being run on the gel, not DNA.

• RNA is not cut with restriction enzyme.
• Special effort is made to prevent the degradation of the RNA by the ever present RNAase.
• Northerns are used to study gene expression.