What the Heck Is PCR?

What The Heck is PCR?

Polymerase chain reaction (PCR) is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested. This technique can be used to identify with a very high-probability, disease-causing viruses and/or bacteria, a deceased person, or a criminal suspect.
In order to use PCR, one must already know the exact sequences which flank (lie on either side of) both ends of a given region of interest in DNA (may be a gene or any sequence). One need not know the DNA sequence in-between. The building-block sequences (nucleotide sequences) of many of the genes and flanking regions of genes of many different organisms are known. We also know that the DNA of different organisms is different (while some genes may be the same, or very similar among organisms, there will always be genes whose DNA sequences differ among different organisms - otherwise, would be the same organism (e.g., same virus, same bacterium, an identical twin; therefore, by identifying the genes which are different, and therefore unique, one can use this information to identify an organism).

Thus, with this amplification potential, there is enough DNA in one-tenth of one-millionth of a liter (0.1 microliter) of human saliva (contains a small number of shed epithelial cells), to use the PCR system to identify a genetic sequence as having come from a human being! Consequently, only a very tiny amount of an organism's DNA need be available originally. Enough DNA is present in an insect trapped within 80 million year-old amber (fossilized pine resin) to amplify by this technique! Scientists have used primers which represent present-day insect's DNA, to do these amplifications.

Here is how PCR is performed:
First step: unknown DNA is heated, which causes the paired strands to separate (single strands now accessible to primers).
Second step: add large excess of primers relative to the amount of DNA being amplified, and cool the reaction mixture to allow double-strands to form again (because of the large excess of primers, the two strands will always bind to the primers, instead of with each other).
Third step: to a mixture of all 4 individual letters (deoxyribonucleotides), add an enzyme which can "read" the opposing strand's "sentence" and extend the primer's "sentence" by "hooking" letters together in the order in which they pair across from one another - A:T and C:G. This particular enzyme is called a DNA polymerase (because makes DNA polymers). One such enzyme used in PCR is called Taq polymerase (originally isolated from a bacterium that can live in hot springs - therefore, can withstand the high temperature necessary for DNA-strand separation, and can be left in the reaction). Now, we have the enzyme synthesizing new DNA in opposite directions - BUT ONLY THIS PARTICULAR REGION OF DNA.

After one cycle, add more primers, add 4-letter mixture, and repeat the cycle. The primers will bind to the "old" sequences as well as to the newly-synthesized sequences. The enzyme will again extend primer sentences ... Finally, there will be PLENTY of DNA - and ALL OF IT will be copies of just this particular region. Therefore, by using different primers which represent flanking regions of different genes of various organisms in SEPARATE experiments, one can determine if in fact, any DNA has been amplified. If it has not, then the primers did not bind to the DNA of the sample, and it is therefore highly unlikely that the DNA of an organism which a given set of primers represents, is present. On the other hand, appearance of DNA by PCR will allow precise identification of the source of the amplified material.

Bacterial Transformation

Introduction:

This is a very basic technique that is used on a daily basis in a molecular biological laboratory. The purpose of this technique is to introduce a foreign plasmid into a bacterium and to use those bacteria to amplify the plasmid in order to make large quantities of it. This is based on the natural function of a plasmid: to transfer genetic information vital to the survival of the bacteria.

The plasmid:

A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base pairs) that contains important genetic information for the growth of bacteria. In nature, this information is often a gene that encodes a protein that will make the bacteria resistant to an antibiotic. Plasmids were discovered in the late sixties, and it was quickly realized that they could be used to amplify a gene of interest. A plasmid containing resistance to an antibiotic (usually ampicillin) is used as a vector. The gene of interest is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli that are sensitive to ampicillin. The bacteria are then spread over a plate that contains ampicillin. The ampicillin provides a selective pressure because only bacteria that have acquired the plasmid can grow on the plate. Therefore, as long as you grow the bacteria in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with your gene of interest that has been inserted to the plasmid.

If you want to get protein made by the bacteria and not just copy the DNA you need to get a copy of the gene with the introns removed. This is necessary because bacteria do not splice introns out of mRNA the way eukaryotes do. This is done using cDNA which is DNA produced by reverse transcriptase from mRNA that is in the cytoplasm. Reverse transcriptase is found in retroviruses, viruses that have RNA as their genetic material. It is used to make DNA from their RNA.

There are many different kinds of plasmids commercially available. All of them contain 1) a selectable marker (i.e., a gene that encodes for antibiotic resistance), 2) an origin of replication (which is used by the DNA making machinery in the bacteria as the starting point to make a copy of the plasmid) and 3) a multiple cloning site. The multiple cloning site has many restriction enzyme sites (to be discussed in a later lab) and is used to insert the DNA of interest. The multiple cloning site is usually in the middle of a reporter gene like Lac Z. A commonly used plasmid is pBluescript:

Figure 1

Competent Cells:Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial cell's membrane. In order to make bacteria take in the plasmid, they must first be made "competent" to take up DNA. This is done by creating small holes in the bacterial cells by suspending them in a solution with a high concentration of calcium. DNA can then be forced into the cells by incubating the cells and the DNA together on ice, placing them briefly at 42oC (heat shock), and then putting them back on ice. This causes the bacteria to take in the DNA. The cells are then plated out on antibiotic containing media.

Competency
The procedure to prepare competent cells can sometimes be tricky. Bacteria aren't very stable when they have holes put in them, and they die easily. A poorly performed procedure can result in cells that aren't very competent to take up DNA. A well- performed procedure will result in very competent cells. The competency of a stock of competent cells is determined by calculating how many E. coli colonies are produced per microgram (10 -6 grams) of DNA added. An excellent preparation of competent cells will give ~108 colonies per ug. A poor preparation will be about 10 4 / ug or less. Our preps should be in the range of 10 5 to 10 6.

In this experiment you will be making competent cells, transforming them with a plasmid and calculating their competency. There will be a lab report due for this lab.

STR Analysis

Restriction enzymes cut DNA at precise points producinga collection of DNA fragments of precisely defined length.These can be separated by electrophoresis, with the smaller fragments migrating farther than the larger fragments. One or more of the fragments can be visualized with a "probe" — a molecule of single-stranded DNA that iscomplementary to a run of nucleotides in one or more of the restriction fragments and is radioactive (or fluorescent).

If probes encounter a complementary sequence of nucleotides in a test sample of DNA, they bind to it by Watson-Crick base pairing and thus identify it.

Polymorphisms are inherited differences found among the individuals in a population.

RFLPs have provided valuable information in many areas of biology, including:

• screening human DNA for the presence of potentially deleterious genes ("Case 1");
• providing evidence to establish the innocence of, or a probability of the guilt of, a crime suspect by DNA "fingerprinting" ("Case 3").

Starting in 1999, law enforcement agencies in both Great Britain and the United States began switching to a new version of RFLP analysis using shorter sequences called STRs ("Short Tandem Repeats").

STRs are repeated sequences of a few (usually four) nucleotides, e.g., TCATTCATTCATTCAT. They often occur in the untranslated parts of known genes (whose sequence can be used for the PCR primers). The exact number of repeats (6, 7, 8, 9, etc.) varies in different people (and, often, in the gene on each chromosome; that is, people are often heterozygous for the marker).

In the U.S., where 13 STRs — scattered over different chromosomes — are examined, the chance that two people picked at random have the same pattern is less than 1 in 1 trillion.

Methods in Analysis of the 13 CODISSTR loci

1. DNA extraction

DNA can be extracted from almost any human tissue. Buccal cells from the inside of the cheek are most commonly used for paternity tests. Sources of DNA found at a crime scene might include blood, semen, tissue from a deceased victim, cells in a hair follicle, and even saliva. DNA extracted from items of evidence is compared to DNA extracted from reference samples from known individuals.

2. PCR Amplification

DNA primers have been optimized to allow amplification of multiple STR loci in a single reaction mixture. By carefully adjusting the distance of the primers from the tetrameric repeat sequence, products from different loci will not overlap during gel electrophoresis.

In the partial results shown to the right, the three STRs D3S1358, vWA, and FGA are being analyzed simultaneously. The lengths of the amplified DNAs are shown by the scale from 100 bp to 280 bp at the top of the figure. The middle panels with multiple peaks are reference standards with the known alleles for each STR locus. Notice that the alleles for the three different loci do not overlap. The lower panel shows the alleles for Bob Blackett's mother Norma for the D3S1358, vWA, and FGA loci. Norma's alleles have been compared by computer to the refrence standards, and labeled.To interpret this result, Norma's genotype is 15, 15 at the locus D3S1358, 14, 16 at vWA, and 24, 25 at FGA.

3. Detection of DNAs after PCR Amplification

The PCR primers in the commercial kits used for STR analysis have fluorescent molecules covalently linked to the primer. To extend the number of different loci that can be analyzed in a single PCR reaction, multiple sets of primers with different "color" fluorescent labels are used. Following the PCR reaction, internal DNA length standards are added to the reaction mixture and the DNAs are separated by length in a capillary gel electrophoresis machine. As DNA peaks elute from the gel they are detected with laser activation. The sequencing machines used for allele separation and detection are the same type currently being used in the Human Genome Sequencing project, with digital output that can be analyzed by special computer software.

In the AmpFLSTR™Profiler Plus™PCR Amplification Kit from Applied Biosystems used by Bob Blackett, 9 STRs are analyzed by using three sets of primers. Each set has a different colored fluorescent label. In the figure above, three sets of STRs are represented by blue, three by green, and three by yellow (shown as black) fluourescent peaks. The red peaks are the DNA size standards. Special computer software is used to display the different colors as separate panels of data and determine the exact length of the DNAs. A tenth marker called AMEL is used to distinguish male DNA as X, Y or female DNA as X, X.

A second kit, called Cofiler Plus, is used in a second PCR reaction to ammplify 4 additional STR loci, plus repeat some of the loci from the Profiler Kit. The result from 2 PCR reactions is the analysis of the entire CODIS set of 13 STRs, with overlap of some loci, and a test for the sex chromosomes. The results are obtained as discrete, digital alleles determined from the exact size of the amplified products compared to known standards.

Cloning animals

Complex biotechnological procedures have enabled scientists to successfully clone mice, sheep, cows and other mammals.

The technology is still at early stages and currently, one in three cloned animals is born abnormally large or with other developmental problems.

The closest scientists have come to cloning a non-human primate occurred in October 2004. Biologists successfully transferred cloned monkey embryos into monkey mothers. None of the resulting pregnancies lasted more than a month.

Somatic cell nuclear transfer

Roslin Institute, Edinburgh

Dolly, the first animal to be cloned, was created using the technique of somatic cell nuclear transfer (SCNT).

To do this, cells are taken from the animal that is going to be cloned. In the case of Dolly the sheep, a cell was taken from normal body cells – somatic cells - in her udder. The nucleus of these cells was removed.

Becasue the nucleus contains all of the genetic material to make the animal, it is termed the donor cell.

Egg cells are used for cloning because of their ability to grow rapidly. The egg cell’s nucleus is removed and the nucleus from the donor cell is inserted in its place.

The egg is then exposed to numerous stimulants which activate the reconstructed embryo, making it divide and grow. The division of the egg cell follows the same process that would occur if the egg was fertilised by sperm during natural reproduction.

The cell division continues for 5 days until a blastomere forms. A blastomere is a ball of nearly 100 cells all with the same genetic material as the donor.

Once a cloned embryo reaches the blastomere stage of development, it can follow two paths. It can be used as a source of stem cells, or it can be implanted into a uterus of a female to create a whole organism. This is called reproductive cloning. When Dolly was born, she was the only lamb born from 277 attempts. She was a clone of the sheep whose udder cell was used.

How To Genetically Modify a Seed, Step By Step

Step one: Finding a new trait

To produce a genetically modified organism, you have to identify the trait you want the plant to have, and find out what other organisms already have it. This involves luck as much as careful searching — Monsanto first produced “Roundup Ready” glyphosate-tolerant plants using a gene from bacteria found growing near a Roundup factory. Ursin pored over science texts outlining organisms’ fatty acid compositions, tested hundreds of flowers and fungi, and finally narrowed down the web of life to two fatty-acid-producing enzymes found in primrose flower and a mold called neurospora.

Concocting a transgenic soybean seed also involves testing the plants themselves to find the most worthy subjects. Monsanto invented some cutting-edge technology to help its scientists make that step more efficient.

Step two: Grabbing genes

In the past, studying the genetic code of individual seeds required planting the seed, growing the plants to a certain size, and then clipping a paper-hole-puncher through a leaf to gather a sample. But that’s a time-consuming and resource-heavy process, so it’s easier to study the seeds themselves, explains Kevin Deppermann, head of Monsanto’s automation engineering department. This requires grinding them up, which is also inconvenient, because a ground-up seed can’t be planted. To get around this, Monsanto engineers invented a special chipping device that shaves off just a tiny piece of the seed and grinds it into a powder that can be analyzed with genome-mapping technology. Meanwhile, the viable remainder of the seed is preserved for planting and cultivation.

Step three: “Trait insertion”

Now that you’ve got your genes, the next step is inserting them into the plants. There are a couple ways to do this, including using “gene guns” that literally shoot pieces of DNA. A .22-caliber charge fires a metal particle coated with DNA into plant tissue. Monsanto no longer uses the technique, but it's still widely used among other biotech companies.

For omega-3 soybeans, Ursin and colleagues used a slightly more delicate process, heating soybean seedlings to place them under stress and make them susceptible to a bug called Agrobacterium tumefaciens. The organism specializes in invading plant DNA and tricking it into producing sugars and amino acids that feed the bacteria. Scientists can exploit this Trojan horse ability and insert new proteins into the plant’s chromosomes. The plant recognizes this foreign encoded protein as one of its own, Ursin said.