Bio 423 Lecture 6

BB211: Cell and Molecular Biology Dr Eve Lutz Department of Bioscience

Recombinant DNA technology: Lecture 6 Polymerase Chain Reaction & Applications Background reading: Reference for this lecture please read the following: Chapter 16 Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.

What is PCR? The polymerase chain reaction (or PCR) is a technique for the in vitro amplification of a desired sequence of DNA. PCR allows the generation of a large quantity of DNA product (up to several µg) from only a few starting copies. It has been shown that PCR can be used to generate a detectable quantity of DNA from only one starting target (or template) molecule. PCR was developed in the mid-1980's, but has already found multiple applications, such as: 1. Rapid amplification of intact genes or gene fragments 2. Generation of large amounts of DNA for sequencing

3. Generation of probes specific for uncloned genes by

selective amplification of a specific segment of cDNA 4. Analysis of mutations for medical applications 5. Detection of minute amounts of DNA for forensic purposes 6. Amplification of chromosomal regions adjacent to genes of known sequence

And many more· Development of PCR won the Nobel prize for Kary Mullis and coworkers.

PCR theory The PCR reaction is a DNA synthesis reaction that depends on the extension of primers annealed to opposite strands of a dsDNA template that has been denatured (melted apart) at temperatures near boiling. By repeating the melting, annealing and extension steps, several copies of the original template DNA can be generated. Promega Amplification AssistantTM website The amount of starting material (target) needed is very small It is not necessary to isolate the desired sequence, because it will be defined by the primers that are used in the reaction. The primers are oligonucleotides complementary to different regions on the 2 strands of DNA template (flanking the region to be amplified). The primer acts as a starting point for DNA synthesis. The oligo is extended from its 3' end by DNA polymerase.

Primer design Oligonucleotide primers used for PCR are usually between 20-30 nucleotides and have ~50% G+C content (melting temperature (Tm) ~55°C to 65°C). See how to calculate Tm. In most situations the primer pair consists of two single oligonucleotides, but degenerate primers can be used if you do not know the exact DNA sequence i.e. only the protein sequence is known or if you are trying to detect other genes in a multigene family - only the 3'end of each primer has to match the target sequence exactly. They are usually designed in order to anneal to regions in the DNA sequence whose distance is ~ 500 bp or less - because short target sequences amplify more easily. If longer fragments are necessary, may need to optimise conditions (amplifying sequences

longer than 10 kb are possible). If the PCR product is to be cloned, then restriction enzyme sites can be incorporated within the 5' ends of the primers. Several primer design computer programmes are available. The stages of a PCR reaction PCR is a cycle of three steps: 1. DENATURATION - the strands of the DNA are melted

apart by heating to 95°C 2. ANNEALING - the temperature is reduced to ~ 55°C to allow the primers to anneal to the target DNA 3. POLYMERISATION - the temperature is changed to the optimum temperature in order for the DNA polymerase to catalyse extension of the primers, i.e. to copy the DNA between the primers. The cycle is repeated over and over again - as many times as needed to produce a detectable amount of product.

Fig 7-38, Lodish et al. (4th ed.)

The principle of PCR existed several years before the technique found widespread use. The first attempts used Klenow fragment of E. coli DNA polymerase I as the enzyme. This enzyme catalyses addition of nucleotides to the 3' end of the primer and has negligible 3' -to5' exonuclease activity. However, the Klenow fragment was not very successful. This enzyme is inactivated by the high temperature (95°C) needed to 'melt' the DNA strands apart and so fresh aliquots had to be added manually for each cycle of the reaction - which is very tedious. In addition, the lower temperatures required for Klenow fragment (37°C) to synthesise the new DNA strand led to the possibility that the primers could anneal non-specifically to the wrong sequences (mismatching primers).

Amplification can occur when mismatching primers are close enough together on opposite strands of DNA - and an unwanted sequence is produced with ends that precisely match the primers. If such an 'incorrect' fragment is synthesised in the early cycles of a PCR, it will be efficiently amplified on subsequent cycles.

Discovery of a thermostable DNA polymerase The breakthrough came with the discovery of the thermostable DNA polymerase Taq polymerase, from the thermophilic bacterium, Thermus aquaticus, which lives in hot springs. Like the Klenow fragment, Taq polymerase does not have a 3' -to- 5' exonuclease activity. However, this enzyme can resist the high temperatures required to melt the template DNA apart without denaturation (loss of activity) and works best at high temperatures (72°C). This led to improved specificity & sensitivity. Annealing of primers to sites other than the target sequence is significantly reduced at the higher temperatures used for Taq polymerase. The fact that only one aliquot ot Taq polymerase needs to be added to the PCR reaction means that all components can be added to the tube at the start of the reaction - allowing the automation of the PCR. Heating blocks which can be programmed to carry out the time and temperature cycles for a PCR have been developed (thermal cycler machine). Template DNA The integrity is more important than the amount (in other words, if the DNA is severely degraded, it is unlikely to have intact target molecules.) Linear DNA is amplified more efficiently than circular, so plasmid DNA should be digested beforehand. Almost any starting material can be used, provided it has not been exposed to nucleases * Can use DNA released by the boiling of cells * Can use DNA from sources which have been preserved or

stored, even for several years o resin/paraffin embedded o dried blood spots o Several thousand year old Egyptian mummies o 10+ thousand year old freeze dried mammoths (well, maybe we're pushing it here) RT-PCR RT-PCR is a reverse transcriptase step combined with PCR amplification and uses RNA as starting material. Typical PCR reaction conditions A typical PCR reaction contains 5 components 1. reaction buffer 2. oligonucleotides 3. template DNA 4. dNTPs 5. enzyme This consists of Tris buffered reaction mixture (pH 8.3) containing 50 mM KCl + 1.5 mM Mg2+ (salt helps primer annealing); between 10 and 50 pmols each oligonucleotide primer (same concentration of each); ~ 1ng of target DNA is sufficient (105-106 molecules); 200 mM each of the 4 dNTPs (dATP, dTTP, dCTP and dGTP); 2 Units of enzyme (1 Unit of activity corresponds to 1 µ mol min-1 reaction rate) Small amounts of neutral detergent and protein (bovine serum albumin) are often added to help stabilise the enzyme. Typical cycling regime Approximately 20-40 cycles of the 3 reaction steps are performed in a PCR reaction. A typical reaction sequence would be: Initial denaturation - 95°C for 2 mins 30 cycles of 95°C (30 seconds denaturation)

55°C (30 seconds primer annealing) 72°C (60 seconds primer extension, for fragments up to 1 kb) Final extension of all DNA ends - 72°C for 10 mins Storage of DNA - 4°C Adjust parameters to suit requirements - If only a small number of target DNA molecules are present in the sample, then more cycles can be performed to generate a detectable amount of product. If the target DNA to be amplified is larger than 1 kb, then longer extension times can be used. •

Calculate time required for extension step as ~1 minute per 1 kb sequence for Taq polymerase

If primers have trouble annealing, can change the Mg2+ concentration or the annealing temperature. * Can adjust Mg2+ concentration between 1-4 mM * Annealing temperature of the primer ~ its Tm, can drop the temperature 1° or 2°C if necessary Specificity can be improved further 'hot-start' method - All the reagents except one are heated to 72°C before adding the final one, usually the enzyme is added afterwards Helps eliminate possible extension from mis-annealed primers at lower temperatures. 'Nested primers' - primers designed to anneal to regions within the target sequence amplified by the first round of PCR. Ensures that only the desired sequence is amplified. Important points to consider to ensure the desired result Is the primer annealing temperature sufficiently high? •

If the primer annealing temperature is too low, non-specific annealing can occur and non-specific products can be formed.

Is Taq polymerase a suitable enzyme? •

Thermostable enzymes which have proof-reading facility (3' -to- 5' exonuclease activity) are now available. Fidelity determines accuracy of PCR amplification

DNA replication is not a perfect process. On occasion DNA polymerases will add an incorrect nucleotide to the growing DNA strand. The rate of misincorporation in a naturally replicating DNA molecule is approximately 1 in 109 nucleotides, this extraordinary accuracy achieved through the proofreading facility of the DNA polymerase.

In vitroTaq polymerase has no 3' -to- 5' exonuclease activity. With temperature and salt concentrations typical of a PCR, the misincorporation rate of ~1 in 104 nucleotides found with using Taq polymerase can introduce a mutation into the target sequence. The enzyme canât distinguish these, which leads to the amplification of sequences which contain the 'mutation' alongside those of the original sequence. Result is mixture of amplified sequences This is less of a problem for many applications. Molecules with the same misincorporated nucleotide will form a very small portion of the total number of molecules synthesised. Misincorporation is important if PCR fragments are to be used for cloning. Since each clone is derived from a single amplified molecule, if the molecule contains one or more misincorporated nucleotides, then all the cloned DNA in that clone will carry the identical mutation. Problem can be reduced by starting with a large, rather than a small number of template molecules. Fewer amplification cycles are needed, resulting in less DNA synthesis.

Contamination can be a major problem

Strictly to be avoided. Contamination occurs when the target DNA is inadvertently introduced into the reaction. This isn't a problem if the only reason for amplifying the DNA is to make more of it, but it is a significant problem if you are using PCR for a diagnostic test. This is why most PCR reactions need to be set up in clean areas with dedicated instruments (such as pipettors), glassware and plastics. DNA is stable, and can be carried through on autoclaved plastics and can even 'fall off your fingertips' skin cells shed from the researcher. The most common source is the products of previous amplifications. These can be carried in aerosol, particularly from pipettors and tips. So be careful.

Applications of PCR 1) Cloning a gene encoding a known protein Primers can be designed from sequence of amino acids or gene sequence. Amplified product can be used as a probe to pull out the full length gene from a cDNA or genomic library 2) Amplifying 'old DNA' Amplifying DNA sequences from museum material or fossils - look at evolution of gene sequences (Molecular evolution studies) 3) Amplifying cloned DNA from vectors Convenient way of checking the inserts - amplified DNA can be analysed by electrophoresis, Southern blotting 4) Creating mutations in cloned genes 5) Rapid amplification of cDNA ends - RACE Most clones in cDNA libraries are not full length. RACE enables the 5' or 3' end of a transcript to be cloned - alternative to rescreening libraries for overlapping clones. Only one gene specific primer is needed. 6) Detecting bacterial or viral infection More sensitive than conventional diagnostic techniques (culturing

samples from patients or using antibodies to detect the presence of microorganisms and viruses) Important for detecting relatively small numbers of organisms * AIDs infection * Tuberculosis (Mycobacterium tuberculosis) 7) Cancer Detecting mutations that occur in cancer and monitoring cancer therapy. Determining if a patient is free of malignant cells 8) Genetic diagnosis a. Diagnosing inherited disorders * Cystic fibrosis * Muscular dystrophy * Haemophilia A and B * Sickle cell anaemia b. Diagnosing cancer - certain cancers are caused by specific and reproducible mutations: e,g. Retinoblastoma - childhood cancer of the eye. The heritable form (germ line mutation of one of the two retinoblastoma allelles): mutation is detected in all cells. Spontaneous form: only detected in tumour tissue. c. Blood group typing d. Prenatal diagnosis - such as determining the sex of foetuses for those at risk of X-linked disorders PCR is one of the most versatile techniques invented, and has so many applications that this list could go on for quite some time. webpage last updated 21/2/03 Back to Recombinant DNA Technology Lecture List