MICROBIOLOGY 101 INTERNET TEXT

TABLE OF CONTENTS

• Introduction
• History of Genetic Engineering
• Palindromic sites
• Restriction Enzymes
• RE Cut Ends
• Blunt Ended Cuts
• Sticky Ended Cuts
• Steps in GE
• Cloning
• Uses of Cloned DNA
• Transgenic Organisms
• DNA Fingerprinting
• Basic Principles of DNA-Fingerprinting
• DNA Separation by Gel Electrophoresis
• How Mutations Effect DNA-Fingerprinting
• Hybridization
• Basic Principle of Hybridization
• Probe DNA
• Polymerase Chain Reaction
• Heat Resistant DNA Polymerase
• Summary of DNA Fingerprinting

What the BASIC TOOLS of GE how are and how they work.

How GENE CLONING is carried out.

How DNA can be used to IDENTIFY all living organisms

How we can DETECT very TINY QUANTITIES of DNA even when they may be millions of years old.

In addition, ETHICAL problems of the GE revolution will be presented and some possible ways of dealing with them considered. 

I have listed some of the changes you may experience within your lifetime, indeed some within the next few years. With the sequencing of the ENTIRE HUMAN GENOME anticipated around the turn of the century, a whole new set of possibilities will be unlocked. Currently (Fall 1996) we have sequenced >10,000 of the ~100,000 human genes, and the rate of sequencing is accelerating as sequencing techniques are improved. About 3,000 human genetic diseases are currently known. This means that approximately 14% of newborns are afflicted with a "visible" genetic disease. This does not begin to include the genetic-based "tendencies" (to develop cancer, arthritis, TB, colds, asthma, flu etc.) and/or "conditions" which all of us have; what genetic conditions do you have? For example, I and my half brother suffer from a genetic condition called Celiac Sprue, which means that wheat protein (gluten) does bad things to the cells in our intestine and we can't eat pizza etc. However, we are FINE as long as we don't eat wheat products. Sprue is a common genetic "condition" that effects up to 25% of the Irish and lessor numbers of other ethnic groups. How many reading this are Irish? Some of you suffer allergies, nearsightedness, migraines, etc., all of which have their basis in your genome. Once the human genome is mapped we can not only identify the particular alleles each of us have, but we will eventually figure a way to replace "undesirable genes" with "good genes". 

One current problem with the genetic revolution is that knowing which gene causes a disease condition and being able to identify the presence of that gene in a person, doesn't mean we UNDERSTAND the molecular biology of the disease process, thus we usually CAN'T PREVENT or CURE the disease caused by a defective gene. An example of this TERRIBLE DILEMMA is that a number of genes that predispose women to breast cancer have been discovered (and new ones are continually being found). This raises important ethical and personal considerations.

If a CURE or PREVENTION is not possible, should a woman be told she carries such a gene?

If a cure or prevention is NOT POSSIBLE, would you want to know you had a time-bomb ticking in you?

If a cure or prevention is not possible, would you consider having your BREASTS REMOVED to prevent getting the disease? If you decide to do this (and many women have), without any sign of cancer, should the Health Insurance Co. pay for the removal of a healthy breast because it is "elective" surgery?

• Let's examine one more "gray-zone" situation before we proceed. Manic depression (MD) is an illness that strikes many people. In some it is relatively mild while in others it takes a terrible toll, often leading to SUICIDE. It is also appears to have a genetic basis as it runs in families, and often stigmatizes affected and unaffected members alike. I would wager money that most of you reading this know someone who is a manic depressive and, of course some of you are MD. Yet, the disease is often treatable by inexpensive drugs and psychotherapy, although many people suffer torment for years before they are properly diagnosed and treated. An interesting side of MD is that many of the creative artists and politicians have been (maybe some of our current politicians are MD) MD. Robert Schumann, Lord Byron, Vincent van Gogh, Winston Churchill and Alfred, Lord Tennyson all suffered the classical symptoms of MD (see "Manic-depressive illness and creativity", Sci. Am. Feb. 1995 & Nov. 1995). Once we identify the genes causing MD, we can use this information to make decisions about whether to have MD gene-positive children or to ABORT any fetus carrying this gene. What would you do? Eventually we will be able to completely cure (repress) the condition. Will the ramification of these findings make for a less interesting and stimulating world? What is your opinion?

Should one's mate, boss, insurance Co., potential employer etc. have access to your genetic information? Can you think of situations where it would be logical and reasonable for others to have this information? How about the army? The CIA?

If you owned a life insurance Co. would you give your political donations to a congressperson who favored requiring potential policy holders to give this information to their insurance Co.

Who pays for the screening for disease-genes, the cost of which currently is high? Should I have to pay for your screening & vice versa?

What if you control the genetic information and you know that giving it out will lead to an abortion; what would you do?

What if you have access to the genetic information of someone your daughter/son, sister/brother etc. is going to marry and you don't think they've told them of a serious genetic "condition". Would you tell? Who owns your loyalty? What is ethical here?

A final reminder of anyone who is considering avoiding these problems; gene therapy trials are currently underway (but they're having problems working: TIME 10/9/95; Science 269:1050 [1995]), people are deciding to abort on the basic of the results of fetal genetic testing and the number of identified defective genes increases almost daily (and some of the ones they find will be ones you and I carry). 

In the 1950s it had been noted that if one grew a particular bacteriophage on a particular bacterial mutant host strain (A) and then infected a bacterial mutant-strain (B), of the same species, with the phage A, the yield of phage from strain B was VERY LOW. However, if you took the few phage B that were produced and infected strain B with them, the phage yield was now NORMAL. Subsequently, in the 1960s, it was found that the phage DNA that grew on strain B had been CHEMICALLY MODIFIED so that it could not be cleaved (destroyed) by a DNase in strain B. The DNase involved in this cleavage was found to be somewhat specific, in that it mainly cut the DNA at CERTAIN SEQUENCES; unless these sequences had been CHEMICALLY MODIFIED by enzymes in the cell (missing in strain A). All previous DNases cut DNA randomly, so this finding suggested that DNA could be cut up into SPECIFIC FRAGMENTS which would ALWAYS contain the SAME SET OF GENES between the cut-sites. However, these first "specific" DNases did not prove as SPECIFIC as hoped. Finally, in 1970 Hamilton Smith accidentally found that a DNase from the bacterium, Haemophilus influenzae, CUT DNA ONLY at UNIQUE DNA SEQUENCES known as PALINDROMIC SITES. 

In DNA a PALINDROMIC SITE is a SEQUENCE OF BASE PAIRS in double stranded DNA that reads the same backwards and forward across the double strand. For example, the sequence of base pairs GAATTC is a palindrome because both sequences of the double strand READ THE SAME when read from either their respective "G" or "C" ends (COMPLEMENTARY strand = CTTAAG). The enzymes that cut these specific sites are calledRESTRICTION ENZYMES (RE). Based on the TYPES OF CUTS they make, there are two types of RE:

One group cuts straight across the double stranded DNA, producing BLUNT ENDED DNA (Fig. 2).

Another type cuts the strand of DNA OFF THE CENTER of the palindromic sites, but between the SAME TWO BASES on the opposite strands. This leaves one or more bases overhanging on each strand and such ends are called STICKY ENDS (Fig. 1) because they form HYDROGEN BONDS with their complementary cut counterparts.

For example, in the base sequence GAATTC, the RE that cuts this site cleaves the DNA between the "G" and the "A" on each complementary strand, thus leaving the overhangs of AATT & TTAA respectively (Fig. 1). Over 200 different RE have been isolated that cut at many different specific DNA sequences. They are all QUITE SPECIFIC and this specificity is the KEY to their use. REs that cut at 4, 5, 6, 8, 9, and 11 base pair palindromic sites have been found. It follows that the MORE BASE PAIRS in the palindromic site the LESS LIKELY a site is to exist statistically. That is, a 4-base-cutting RE cuts the same DNA many more times than an 8-base-cutter does. The odd numbered cutting sites are not true DNA palindromes, in that the central bp reads different in the two directions. For example AA?TT is a 5 bp cutting site, where the ? = a number of different bases.
 

The final important component in genetic engineering is an enzyme called LIGASE. Ligase is an enzyme that covalently joins the sugar-phosphate backbone of bases together. Ligase is also involved in DNA replication. In effect LIGASE reverses the action of the RE, which breaks the sugar-phosphate backbone-bonds. Ligase requires energy to form these bonds (Fig. 3). Ligase will join either "sticky" ends or "blunt" ends, but it is more efficient at closing sticky ends because the "sticky overhangs" of these ends adds stability which holds the stands together while the ligase works, whereas the blunt ends are LESS STABLE. The sticky ends must have the SAME "base overhang" (Fig. 1), but ANY BLUNT END can be joined to any other blunt end (Fig. 2).
 

Review of the components of GE:

(1) Double-stranded DNA contain palindromic restriction enzyme sites.

(2) Many types of restriction enzymes cut or cleave palindromic sites in the double-stranded DNA sample forming either a sticky or a blunt end.

(3) The enzyme ligase plus an energy source fuses or join two sticky or two blunt ends of DNA together. 

Isolating the SOURCE and VECTOR DNA. The DNAs must be relatively free of contaminating materials which interfere with the subsequent enzymatic steps.

Both the source and vector DNAs are cut with RESTRICTION ENZYMES. When sticky ends are formed the DNA is cut with the same restriction enzyme(s), but RE that produce blunts ends also work well in cloning.

The vector and source DNAs are mixed with a LIGASE SYSTEM and covalently bond together.

Finally, the LIGATED DNA is TRANSFORMED into a host cell. Usually the host cell is a COMPETENT bacterium, but increasingly eukaryotic cells are being used. After suitable growth has occurred the host cells are examined for the PRESENCE of the source or CLONED DNA in its cytoplasm.

The entire cloning process often takes LESS THAN A DAY and is carried out using volumes of 1,000 microliters or less. The examination of the transformed host for the gene-of-interest may take an additional day or so. A gene which has been successfully transferred in this way is said to have been CLONED. The process is referred to as "CLONING", "RECOMBINANT DNA TECHNOLOGY", or "RECOMBINANT DNA". These steps are summarized in the figure 4 below.
 

SEQUENCING: Sequencing determines the base pair sequence of a gene. By reading the 3-letter code, sequencing also describes the AMINO ACID SEQUENCE translated from that gene.

MUTATION: The gene bp sequence can be changed in specific ways and the modified gene can be inserted back into its original host to see what each specific mutation does. Whereas, spontaneous mutation is random, techniques are now available that make it possible to CHANGE any codon within a gene to any other codon. Therefore, it is possible to study the effects of SINGLE AMINO ACID CHANGES on the function of the gene product, which is, after all, the ultimate purpose of the exercise.

To replace a mutated form of the gene in the original host cell with a healthy form of the gene to see exactly what it does in its intended place of residence.

To use the amplified gene to make HUMONGOUS QUANTITIES of the GENE PRODUCT for commercial purposes. This is how products like human insulin, human growth hormone, plasminogen activator, interlukins and the bovine milk hormone are all produced.

To INSERT the gene into ANOTHER SPECIES for some purpose. Such animals or plants are said to be TRANSGENIC. For example, many crops are protected against caterpillar larvae because a gene from a bacterium has been inserted into their cells. This gene produces a protein that is HIGHLY, and SPECIFICALLY, TOXIC to the larvae of many moths and butterflies that attack food crops. 

There is a DOWN SIDE of GE. The same procedures that can be used to produce a better crop, or human insulin, can also be employed to make a more POWERFUL PATHOGEN. There is evidence that some countries have or are considering doing this. For example, Iraq was apparently growing bacterial pathogens for biological warfare, but it was deterred from using them when we threatened atomic retaliation. It is only a small jump in "reasoning" to consider engineering a "better pathogen" with which to destroy a hated enemy (fill in blank--the news report that some Americans. consider anyone working for the government to be the enemy). For more information read "The spectra of biological weapons" Scientific Am. Dec. 1996, pg. 60.
 

which I only watched because of my scientific interest, of course).

The THREE basic principles required to understand DNA fingerprinting and all that follows from it you already know (the principle of ligand/receptor binding; see fig.2 chap 7), but it is worth taking a moment to review them.

BASE-PAIRING of AT (AU) & GC is the BASIC PRINCIPLE of this procedure. If you understand this all else fall easily into place.

SPECIFICITY of enzyme activity is the second CRUCIAL principle to understanding DNA fingerprinting. This refers to the cutting of DNA by specific RESTRICTION ENZYMES at UNIQUE palindromic sequences.

The recognition that a CHANGE in a SINGLE BASE PAIR (a mutation) can either MAKE a RE-site where one did not exist previously or it can REMOVE or ELIMINATE a RE site from a gene. An analogy would be to "mutate" your phone number by one letter; callers would get a different person.

To understand DNA fingerprinting the double stranded DNA molecule must be viewed as a chain with RANDOM RESTRICTION ENZYMES-sites (palindromes) located along its length. If each of these various unique restriction sites were marked with a different color, the DNA would appear as a random multicolored stripped ribbon. If one set of unique restriction enzyme sites were colored RED and the appropriate RED-restriction enzyme added, the DNA would be CLEAVED into a series of VARIOUS SIZED FRAGMENTS depending on where the RED-SITES were located along the DNA molecule (Fig. 6). It is easy to understand that the group of DIFFERENT SIZED FRAGMENTS would be UNIQUE for two DNA strands with the RED-restriction sites located at DIFFERENT PLACES along their respective DNA double strands. The addition of a GREEN-restriction enzyme that cut GREEN-SITES would produce a different group of sized fragments. Each of these groups of restriction enzyme-produced fragments would represent a unique FINGERPRINT of that DNA (Fig. 6).
 

The DNA fragments are visualized using the technique known as GEL ELECTROPHORESIS, which you performed in lab exercise 15. Briefly, porous gels composed either of a PLASTIC called ACRYLAMIDE or of a derivative of agar, called AGAROSE, are used to separate DNA fragments based on size. The gels are prepared with wells into which the DNA fragments are ADDED. The gels are submerged, under an electrolyte buffer solution between a positive and a negative electrode; the DNA-containing solutions are added to the wells and the current is turned on. The DNA fragments are NEGATIVELY CHARGED so the wells containing them are placed closest to the negative electrode. When the current is turned on the DNA moves through the pores in the gel TOWARDS THE POSITIVE ELECTRODE. The shorter fragments move FASTEST because they are able to navigate through the pores of the gel more easily, whereas the longer DNA fragments move PROPORTIONALLY MORE SLOWLY through the pores. The result is a separation of the DNA fragments based on their length (SIZE). The DNAs are stained by dyes that binds to the DNA and fluoresces strongly when exposed to UV-light. The various sized DNA fragments appears as a series of bands that resemble a BAR CODE. See Fig. 13. 

In Figure 7 the gel pattern of a hypothetical analysis of O.J.'s DNA vs. a sample of unknown DNA is depicted. The two isolated DNAs were treated with the same RE and the fragments separated on an agarose gel. Two unique restriction enzyme fragment patterns are seen. The conclusion is that the blood samples came from DIFFERENT INDIVIDUALS. The SOURCE of the DNAs clearly influences the significance of these results. For example, if the two samples were collected at the scene of the crime it would mean one thing, but if the unknown DNA sample came from O.J.'s shirt and it matched that of one of the victims at the scene of the crime, the data takes on more significance.
 

ANSWER: This is exactly what happens. When genomic DNA is treated with a RE and separated on a gel, what is seen is a continuous smear composed of 1,000s of individual DNA fragments. This problem is solved by a technique known as HYBRIDIZATION.

Hybridization again depends on the basic principle of HYDROGEN BONDING between GC & AT bonds in nucleic acids. The principles in the hybridization steps are:

DNA strands are SEPARATED by breaking the hydrogen bonds between the bases with heat to produce SINGLE STRANDS.

Strand separation is STRICTLY DEPENDENT up on two factors: The TEMPERATURE (amount of heat energy) and the NUMBER of hydrogen bonds (an incorrect base pairing does NOT form H-bonds).

When the temperature drops, single DNA strands in the same solution that are complementary SPONTANEOUSLY COME TOGETHER by pairing up through their respective AT & GC associations. The strength of their subsequent association depends on the temperature and the total number of MATCHING base pairs.

The TEST or SAMPLE DNA is usually FIXED or BOUND to a solid surface in a SINGLE-STRANDED STATE. A piece of DNA of KNOWN SEQUENCE to which something is attached that can be DETECTED or SEEN is then mixed with the bound DNA and the two are incubated together under conditions that will allow COMPLEMENTARY DNA sequences to join through the appropriate hydrogen bonding. The DNA molecule which can be detected is called a PROBE.
 

That is, the MORE hydrogen bonds there are the HIGHER the temperature must be to completely SEPARATE two DNA strands and to keep them separated. For example, if there were two stands of DNA composed of 200 PERFECTLY complemented base pairs, then it would take a temperature of 58^oC. to separate them. However, if only 199 of the base pairs were correct, it would only take 57^oC. to separate them; if there were only 100 correct base pairs the DNA strands would fall apart at ~29^oC; i.e., fewer bonds require less heat (energy = lower temperatures) to rupture them.
 

Hybridization is carried out as illustrated in Fig. 10:

1. The isolated DNA is FRAGMENTED (CUT) with restriction enzymes.
2. The cut-DNA is separated into fragments according to SIZE on a gel.
3. The DNA fragments are transferred, IN POSITION, to a membrane to which they TIGHTLY BIND.
4. The membrane-bound-DNA is soaked in a solution containing PROBE-DNA that is labeled with something that can be DETECTED (seen by the eye).
5. The membrane-bound-DNA-probe mixture is incubated at a temperature designed so that ONLY THE PROBE DNA strands that have a high degree of COMPLEMENTARITY (base pair matching) to DNA strands bound to the membrane will be able to bind (Hydrogen-bond) to each other.
6. The unbound probe is washed away and the position of the bound probe is determined using its detection system of the probe.

Figure 10. The hybridization procedure.

The entire procedure is called HYBRIDIZATION. Hybridization works because of the SEQUENCE OF THE PROBE. A given probe will only bind with DNA that is attached to the membrane IF IT FINDS a MATCHING or COMPLEMENTARY DNA base pair sequence that forms enough bonds to remain together under the heating conditions used. Thus if no matching complementary sequences are found, NO PROBE will bind (Fig. 10, green DNA). If one or two of the 1,000s of the bound DNA fragments contain a sequence complementary to the sequence in the probe, the probe will BIND only to these fragments and thus become ATTACHED TO THE MEMBRANE through this association with the complementary membrane-bound-DNA (Fig. 10, red DNA). It is possible to chose probes that are known to bind to specific sequences or artificial probes of KNOWN SEQUENCES can be made, for about $1.00/base and sent to you in 48 hr.; THEY CAN BE ORDERED OVER THE INTERNET.
 

In Fig. 12 three different PROBES (A, B, & C) are hybridized against the separated membrane-bound-DNA fragments shown on the right (from 3 duplicate gels). As an exercise determine WHICH BANDS each of the three probes will hybridize (bind) to; that is, draw three duplicate gels, 1, 2, & 3 and circle the band(s) in gel 1 that probe A will bind to; in gels 2 & 3 do the same with probes B & C respectively.
 

Figure 14 illustrates how the blood from O.J.'s trial was tested to determine matches. The DNA from the various samples of blood (from the gloves, the Bronco, the socks, the bodies etc.) was extracted, digested with one or more restriction enzymes, separated on gels, transferred to DNA-binding membranes and hybridized with various probes. The pattern of the DNA fragments (bands) from the samples that "lights up" were then compared for similarities and differences.
 

FAQ: About DNA fingerprinting.

1 How good (accurate) is it at identification. For example, is it as good as classical fingerprints?

Answer: In theory, with the exception of identical twins, EVERYONE on this planet has a different DNA fingerprint. That is, DNA fingerprinting IS as good (distinctive) as classical fingerprinting for identification.

2. What are its advantages?

Answer: In theory DNA fingerprinting will work with much smaller amounts of material than a classical fingerprint & DNA lasts much longer than classical fingerprints. DNA-containing samples that are many years old (up to 25 million yr.) are still usable. Only very tiny quantities of DNA are required in order to carry out a highly accurate test. For example, dried blood, semen, spit, skin etc. on samples stored in dusty files for years are still usable. Samples of mixed DNA's can also be used. DNA containing evidence is much harder to clean up at a crime scene than other evidence, like classical fingerprints.

3. What are its limitations?

Answer: There currently are no accepted Federal standards for controlling the quality of DNA testing nationwide. Poor quality & poorly controlled testing leads to QUESTIONABLE and SHODDY RESULTS. Lab A may use one set of procedures and standards, whereas, lab B may use another set of procedures and standards. Making comparisons between results from the two labs difficult. The quality of laboratory personal is not standardized. For example, clinical laboratory technicians, that work in hospitals and clinical labs, must be certified by their states and by a national organization that sets high standards of training and experience. The clinical labs are frequently tested to determine if their procedures are done correctly. They receive & must analyze test samples whose composition is known by certification agencies. The results are returned to the certification agencies for evaluation. If a clinical lab makes too many errors the lab will be DECERTIFIED. This is not yet the case with DNA testing facilities.
 

Even if there is a perfect match between DNAs, you can not say HOW the DNA containing sample got there or WHEN. In the O.J. trial a VALID question was raised about the possibility of evidence being planted. What makes this charge so powerful is the EXTREME SENSITIVITY of the procedure. That is, it would NOT BE DIFFICULT for someone to collect a small quantity of blood (one or two drops from a tube of a blood sample), body fluid or tissue from a victim (e.g. hair in a comb, blood on a Kleenex etc. ) and place this material where it would incriminate an innocent person. There are documented cases where quantities of drugs, marked money etc. have been "planted" and innocent people convicted of crimes because of these actions.

Finally, blood that is mixed with the wrong chemicals or is degraded is difficult to analyze accurately.

4. When faced with DNA evidence what questions should be asked?

Answer: As indicated above (and in the O.J. trial), questions regarding the handling of the evidence, the quality of the testing, including the quality controls used, the skill of the testing personnel, the accuracy of the data interpretation, possible contamination of the evidence, the possibility of accidental, or intentional misplacement of evidence are all valid questions that should be raised regarding DNA evidence (or any evidence for that matter). As the sensitivity of this powerful technique improves and its use widens throughout our justice system, it is important that we deal with these problems if we expect EQUAL JUSTICE for all. We must not lose sight of the fact that no matter how powerful a new tool in crime fighting is, its ultimate effectiveness is only as good as the persons using that tool. 

The PCR is another one of these discoveries, like that of the structure of DNA, the transforming factor determining the chemical nature of DNA, and restriction enzymes, that generated a quantum leap in science. Almost the instant after PCR is explained to a biological scientist, ideas as to its uses begin to pour from all but the most dull scientific mind. Even today, years after its discovery, we are still developing new uses for PCR.

The irony of the PCR is that living organisms have been doing it since they evolved in the primeval organic soup of this planet 3.5 billion years ago and scientists have been aware of the general DETAILS of this process for ~50 years. To put it succinctly, the PCR does in the test tube what every bacterium does in its tube of media or on an agar-plate and each of us do every day; we all produce billions of exact copies of our own DNA; AMPLIFYING our DNA millions of time. The enzyme DNA polymerase was discovered in the 1950s and our knowledge of the process has been increasing ever since. This means that thousands of scientists have studied DNA replication for 40 years without tumbling to PCR.

The basic principle of PCR is shown in Fig. 15. It has been know for a long time that DNA polymerase requires a short stand of DNA or RNA called a PRIMER to "prime" the START OF DNA REPLICATION. Mullis's genius was that he reasoned "That if you added the following components to a test tube containing a single DNA molecule, you could replicate & amplify that DNA molecule many million fold in a short time.

The components are:

A sample of the TARGET DNA to be copied. In theory only a single molecule is needed.

A set of short (15 to 40 bases) single stranded PRIMERS of DNA, in EXCESS, that will bind to complementary regions of the opposing stands of the TARGET DNA molecule . These primers BRACKET the region of DNA to be amplified.

An EXCESS of the 4 nucleotide triphosphates, ATP, GTP, CTP, TTP.

The enzyme, DNA polymerase.

Various buffers and cofactors like magnesium ions required by DNA polymerase.

The final trick was to get the two target DNA stands APART (separated) so the primers could bind and the DNA polymerase could do its thing. It had been known for ~50 years that heat separates DNA stands and that complementary strands then rejoin through base pairing when the temperature is subsequently lowered. So Mullis heated his mixture of target DNA, primers and triphosphate nucleotides to about 90^oC for a few minutes to separate the target DNA. He then lowered the temperature enough to allow the primers, which were small and in VAST EXCESS, to bind (ANNEAL) to their respective complementary target DNA bp-sequences. At this point he added DNA polymerase and allowed the polymerization reaction with the triphosphate nucleotides to occur. That is, the DNA polymerase FILLED IN the missing portion of each strand making TWO NEW DOUBLE STRANDED regions of DNA.
 

Each entire PCR cycle takes only 2 to 10 minutes. However, there was one problem with the system devised by K. Mullis, which was that the DNA polymerase he used was DESTROYED BY THE HEATING process. Mullis had to add new DNA polymerase for EACH ROUND, which was time-consuming and expensive. This problem was solved by using HEAT-RESISTANT DNA polymerase that only needed to be added once. THERMOPHILIC bacteria that live in boiling hot springs have been described previously. This is another case of SERENDIPITOUS BASIC SCIENCE turning out to be important. The study of thermophiles might seem to be a waste of money and time to many people as these bacteria, while certainly interesting, don't cause disease in man or any other life form. The argument could be made that a scientist could better spend his/her time working on cancer or some other terrible disease that afflicts humankind. However, it turns out that since thermophilic DNA polymerases tolerate high temperatures (e.g. 90^oC) for long periods without being destroyed, they are the perfect solution to the PCR DNA polymerase problem. Thus today PCR has become a revolutionary tool because of scientists who studied these odd thermophiles.
 

The standard PCR reaction is run through about 30 cycles in a couple of hours which results in the amplification of the original DNA by over a 10⁹ fold. Thus a single specific DNA region can be amplified to yield sufficient quantities to do anything that can be done with bulk-isolated DNA. In the case of crime evidence this means that the DNA in a single hair follicle, a single drop of semen or blood is sufficient to prove that an individual was present at the scene of a crime. Indeed any piece of evidence that contains one or more moderately long DNA fragments (e.g. >200 base pairs) can be amplified with PCR and its RFLP determined for identification purposes---or maybe to build a dinosaur eventually. However, at present the maximum limit for amplification is approximately 42 kilobases. 

The major limitations of DNA fingerprinting are human error and the quality of the technology used.

The discovery of PCR increased the sensitivity and versatility of DNA fingerprinting, and changed the face of molecular biology.

These techniques will allow infectious diseases to be identified within hours or even minutes. They are accelerating the rate of sequencing the human genome, they allow the investigation of complex ecological interactions and species relationships that here-to-fore have been too complex to study.
 

Copyright © Dr. R. E. Hurlbert, 1996. This material may be used for educational purposes only and may not be duplicated for commercial purposes.