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Electrophoretic Analysis of DNA   **Practice Plasmid.exe -- It will be on the Quiz**
Cloning genes
PCR
Gene sequence analysis
Complementarity and Hybridization
Northern, Southern, and Western blots
DNA Microarrays
Transgenics


What is gene technology?  All gene manipulation is based on microbial genetics--ways of doing in the test tube what bacteria and viruses do naturally. Several of this week's gene manipulations are exemplified by the article on overexpressed angiopoietin in a transgenic mouse.

Gel Electrophoresis Images and text based on MIT Hypertextbook

This technique separates molecules on the basis of their size.
  The gel is set up for electrophoresis in a tank holding pH buffer.   Electrodes apply an electric field:


MIT hypertextbook

The molecules to separate (DNA RNA) carry a net negative charge (why?) so  they move along the electric field toward the positive cathode.   (To separate proteins, a detergent would be included which coats the protein with negative charge.)

The larger molecules are held up as they try to pass through the pores of the gel, while the smaller molecules are impeded less and move faster.  This results in separation by size, with the larger molecules nearer the well and the smaller molecules farther away.

Note that this separates on the basis of size (volume in solution), which is not necessarily molecular weight. For example:

Aside from the above exceptions, the distance migrated is roughly proportional to the log of the inverse of the molecular weight (the log of 1/MW). Gels are normally depicted as running vertically, with the wells at the top and the direction of migration downwards. This leaves the large molecules at the top and the smaller molecules at the bottom. Molecular weights are measured with different units for DNA, RNA, and protein: Molecular weight standards run in one well of the gel are used to calibrate the molecular weights of sample molecules.  Below is a gel stained with a dye: a colored molecule which binds to a specific class of macromolecules in a sequence-independent manner (probes bind in a sequence-dependent manner).

Sample 1 contains only one size class of macromolecule - it could be a plasmid, a pure mRNA transcript, or a purified protein. In this case, you would not have to use a probe to detect the molecule of interest since there is only one type of molecule present. Blotting is usually necessary for samples that are not complex mixtures. By interpolation, its molecular weight is roughly 3.

Sample 2 is what a sample of total DNA cut with a restriction enzyme, total cellular RNA, or total cellular protein would look like in a gel stained with a sequence-independent stain. There are so many bands that it is impossible to find the one we are interested in. Without a probe (which acts like a sequence-dependent stain) we cannot get very much information from a sample like this.


MIT hypertextbook

Different stains  are used for different classes of macromolecules.  DNA and RNA are generally stained with ethidium bromide (EtBr), an intercalating agent.  The DNA-EtBr complex fluoresces under UV light.   Protein is stained with Coomassie Blue or Silver Stain.


Cloning genes
In nature, DNA molecules recombine for various functions -- even DNA between different species.  But twenty years ago, despite the work of Barbara McClintock and others, the extent of this recombination was not appreciated.  DNA was still thought to be the "master molecule," not to be violated by "unnatural" manipulation.  When scientists began to manipulate DNA in the test tube,  many scientists feared that disastrous monsters would result, with unspecified dangers to people.  In 1977 scientists at the Asilomar Conference proposed sweeping regulation on so-called "recombinant DNA," technologies which recombine DNA from different species in the test tube.

Since then, the dangers have appeared to be little  more than those of "natural" genetic mixing.  But we remain concerned about issues such as:

Techniques of Recombinant DNA
How do we manipulate these natural processes for biotechnology; for instance, to make a bacterium that produces large quantities of insulin?

One approach would be to cut the appropriate gene from human DNA and paste, or splice, it into a vector such as a plasmid or phage DNA.  Our "scissors" are the class of enzymes called restriction endonucleases

Restriction Endonucleases
An "endonuclease" is an enzyme that cuts duplex DNA in the middle, not at an end (for exonuclease).   Different species of bacteria have evolved different restriction endonucleases, each to cut foreign DNA that gets into their cells by mistake.  To be cut, the DNA has to lack their own pattern of protective methylation.   There are well over a hundred restriction enzymes, each cutting in a very precise way a specific base sequence of the DNA molecule.

A restriction endonuclease cuts DNA only at a specific site, usually containing 4-6 base pairs.  The enzyme has to cut the DNA backbone twice, recognizing the same type of site; therefore, the site "reads" the same way backwards as forwards--a palindrome.

This "sticky ends" from two different DNA molecules can hybridize together; then the nicks are sealed using ligase.  (Where does ligase come from?  What is its natural function?) The result is recombinant DNA.  When this recombinant vector is inserted into E. coli, the cell will be able to process the instructions to assemble the amino acids for insulin production. More importantly, the new instructions are passed along to the next generation of E. coli cells in the process known as gene cloning.








Restriction site Analysis
How can we use restriction sites to analyze the plasmid products of ligation, and tell whether we in fact have ligated the correct molecule:
Problem: Suggest several "incorrect" ways the plasmid could recombine.

More Problems:
Use the PLASMID Program.  You MUST practice restriction analysis with this program; it will be on the quiz and/or the test.

How do we get the recombinant molecule into a bacterial cell?  Usually by transformation (for a plasmid) or by in vitro packaging into a phage head coat (for a phage vector such as lambda phage).

The above is a highly simplified description of  recombinant DNA technology.
How would we actually locate the appropriately cloned gene?  There are many different ways, depending on the specific case.  Here is one example, in which a partial sequence of the protein enables us to reverse the code and determine an approximate DNA sequence to use for a radiolabeled probe.  The DNA probe will hybridize to clones containing the correct DNA, even if it is just one piece cut out of an entire genome.



Griffiths et al, W. H. Freeman & Co., current edition

The radioactive probe is made by determining a short segment of the protein sequence, then "back translating" to the possible DNA sequences.  Short DNA sequences are synthesized to match the  protein sequence.  Then these DNA oligomers (known as "oligos") are radiolabeled, and applied to the blotted clones.  They should hybridize only to clones containing sequence encoding the desired protein.

Reverse transcription: cDNA Cloning
Suppose we need to clone a gene containing lots of introns.  What will happen when the bacterium tries to express it?
To overcome this problem, we can start with mRNA isolated  from tissues that produce the desired protein.  We then use reverse transcriptase enzyme (produced by a retrovirus related to HIV) to reverse transcribe the mRNA into a DNA molecule that now is free of introns.  Now we can ligate "sticky ends" onto the cDNA and recombine it into a phage or plasmid vector.

Problem:  Know the differences between genomic cloning and cDNA cloning.
Explain the relative ADVANTAGES and DISADVANTAGES of each technique--depending on the aim of your research.

PCR: Summary of technique

In PCR, a heat-stable DNA polymerase is used, most commonly Taq Polymerase from the thermophilic microbe Thermus aquaticus.  Thomas Brock discovered T. aquaticus  from a hot spring at Yellowstone National Park.

Prismatic Pool, Yellowstone

More recently, an even more heat-resistant polymerase has been developed from a hyperthermophilic microbe growing at 110 degrees C in hydrothermal vent ecosystems in the deep ocean; it's called "Vent Polymerase."

The Taq Polymerase is put with the DNA to be amplified, plus all four NTPs, plus two primers facing each other, about 200 - 6000 kb apart.  (Why do we need primers?)  The primers are selected based on the DNA region you want to amplify.  The tube is placed in a thermal cycler.

DNA gets synthesized from each primer, for about 2 minutes.  Then the temperature is raised to 95 degrees C -- enough to denature (split apart) the DNA base pairs.  But the Taq Polymerase remains intact, because it comes from an organism that evolved to grow at this temperature.

Now the temperature is decreased again, and primers again can hybridize to the DNA--both the old AND the newly synthesized strands.  Again, Taq Polymerase extends new DNA strands.  Again, the temperature is raised.

After repeated cycles, the amount of DNA sequence between the two primers increases exponentially.  First 2 strands, then 4, 8, 16, up to about a million.  Thus, in a couple of hours, you can get million-fold amplification of a DNA sequence.



Griffiths et al, W. H. Freeman & Co., current edition









Applications of PCR
PCR has replaced cloning for many purposes, particularly the sequencing of DNA.  It is faster and requires no vectors, which can mutate as they reproduce.   It can be used forensically, to amplify tiny amounts of DNA from criminal evidence; or clinically, to detect DNA sequences linked to inherited disorders.
 
 
 

PCR Experiment in 
Microbiology Lab:

Students characterized the microbial species of various environments in Knox County.

Microbial colonies were placed directly into PCR reactions containing primers for amplification of ribosomal RNA genes (rDNA).

Lane 4 contains an amplified band of the predicted size, 1000 bp.  The top of each well contains genomic DNA.  The smears at the bottom contain the PCR primers.

The DNA will be sequenced and matched through GenBank to determine the microbial genus.

Thanks to Dan Nickerson '00 and Adam Marks '01.

The main limitations of PCR are:

For more information on PCR see:

Gene sequence analysis  Modified from MIT Hypertextbook.

The sequence of DNA base pairs can be analyzed by


Once we have a piece of DNA cloned, it is amplified (available in many copies) and we now have a living clone which provides, in theory, an indefinite source of the DNA sequence.  How do we analyze the sequence?

DNA Sequence Determination
Once you have identified a particular region of DNA of interest, you need to find out the precise sequence of DNA nucleotides.  This is done by di-deoxy sequencing, in which a DNA polymerase is put together with dNTPs in four different reactions, each containing a small amount of one di-deoxy NTP (ATP, TTP, CTP, or GTP).  The di-deoxy nucleotide lacks a 3'OH to continue chain extension, so the chain terminates.  Each reaction produces a population of fragments terminated at A, T, C, or G.

  • The fragments are either radiolabeled or enzymatically labeled.  They can be separated on a gel, or on a fluorescence analyzer.  All published DNA sequences in the world are deposited in GenBank.
     


     





     
     
     

    images from Davidson College

    Complementarity and Hybridization
    How do we find genes of interest in a genome?  Or a particular mRNA in the total cellular RNA?   Or a particular protein out of all cell protein?

    In solution, hybrid molecular complexes (usually called hybrids) of the following types can exist (other combinations are possible):

    There are two important features of hybridization: These properties allow you to use hybridization to perform a molecular search for one DNA molecule, or one RNA molecule, or one protein molecule in a complex mixture containing many similar molecules.
    Southern, Northern, and Western Blots.   Link to summary.

    Blots are named for the target molecule.

    Southern Blot--DNA cut with restriction enzymes - probed with radioactive DNA.
    Northern Blot--RNA - probed with radioactive DNA or RNA.
                            Example--used to measure angiopoietin angiopoietin expression from cDNA in transgenic mouse.
    Western Blot--Protein - probed with radioactive or enzymatically-tagged antibodies.

    These molecules must then be immobilized on a solid support, so that they will remain in position during probing and washing. The probe is then added, the non-specifically bound probe is removed, and the probe is detected. The place where the probe is detected corresponds to the location of the immobilized target molecule. This process is diagrammed below:

    In the case of Southern, Northern, and Western blots, the initial separation of molecules is done on the basis of molecular weight, by gel electrophoresis.

    Preparing for Blots
    • Southern Blots.  DNA is first cut with restriction enzymes and the resulting double-stranded DNA fragments have an extended rod conformation without pre-treatment.
    • Northern Blots.  Although RNA is single-stranded, RNA molecules often have small regions that can form base-paired secondary structures. To prevent this, the RNA is pre-treated with formaldehyde.
    • Western Blots.  Proteins have extensive 2' and 3' structures and are not always negatively charged. Proteins are treated with the detergent SDS (sodium dodecyl sulfate) which removes 2' and 3' structure and coats the protein with negative charges.

    Transfer to Solid Support.  After the DNA, RNA, or protein has been separated by molecular weight, it must be transferred to a solid support before hybridization. (Hybridization does not work well in a gel.) This transfer process is called blotting and is why these hybridization techniques are called blots. Usually, the solid support is a sheet of nitrocellulose paper (sometimes called a filter because the sheets of nitrocellulose were originally used as filter paper), although other materials are sometimes used. DNA, RNA, and protein stick well to nitrocellulose in a sequence-independent manner.

    After a series of treatment steps, the probe is added.   The probe hybridized to the target molecules is visualized either by autoradiography or by enzyme reaction.

    Summary.  The important properties of the three blotting procedures of DNA analysis:

    DNA Microarrays

    We can now put most of the protein-encoding genes onto a microarray chip, using technology based on the DNA silicon chip industry.  The chip can be used to hybridize to cellular RNA, and measure the expression rates of a large number of genes in a cell.  More details here.


    Axon Industries.
    From "Everything's Great When It Sits on a Chip," The Scientist,  Volume 13, #11, May 24, 1999

    Transgenics

    Cloning in Animals.
    Animals have two different classes of cells: germ line and somatic.
    Only alterations in the germ cells can be transmitted to future generations.  However, some forms of somatic cell gene therapy can be useful in treating patients.  For example, people with cystic fibrosis can receive the cloned CFTR gene in a nasal spray, which then infects the lining of their lungs and improves lung function.  The infected (transformed) epithelial cells eventually are lost, however, during normal processes of tissue growth, so the treatment needs to be repeated.

    Germ-line cloning.  There are two basic ways to clone a gene in the mammalian germ line.

    Once you have cloned a valuable gene into an animal, say a sheep that makes insulin in its milk, how can we produce lots of identical progeny quickly without sexual reproduction?   This is what the popular press calls "cloning."   (More after spring break.)

    But when you create a clone, how do you know it worked?  You need to know:

    Cloning in Plants.
    Plants don't have separate somatic and germ cells, so creating transgenic plants is easier than creating transgenic animals. Techniques include the use of microprojectile bombardment (the "gene gun") and the use of Agrobacterium tumefaciens.

    Online practice problems: Try problems 1-9 of the Recombinant DNA Technology Problem Set from the Biology Project at the Univerisity of Arizona.

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