Introduction • PCR, polymerase chain reaction, is an in-vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence – PCR is a mean to amplify a particular piece of DNA • Amplify= making numerous copies of a segment of DNA • PCR can make billions of copies of a target sequence of DNA in a few hours • PCR was invented in the 1984 as a way to make numerous copies of DNA fragments in the laboratory • Its applications are vast and PCR is now an integral part of Molecular Biology
• 1966, Thomas Brock discovered Thermus Aquaticus, a thermostable bacteria in the hot springs of Yellowstone National Park • 1983, Kary Mullis postulated the concept of PCR ( Nobel Prize in 1993) • 1985, Saiki published the first application of PCR ( beta-Globin) • 1985, Cetus Corp. Scientists isolated Thermostable Taq Polymerase (from T.Aquaticus), which revolutionized PCR
DNA Replication vs. PCR • PCR is a laboratory version of DNA Replication in cells • The laboratory version is commonly called “ in vitro ” since it occurs in a test tube while “ in vivo ” signifies occurring in a living cell.
DNA Replication in Cells ( in vivo ) • DNA replication is the copying of DNA • It typically takes a cell just a few hours to copy all of its DNA • DNA replication is semi-conservative (i.e. one strand of the DNA is used as the template for the growth of a new DNA strand) • This process occurs with very few errors (on average there is one error per 1 billion nucleotides copied) • More than a dozen enzymes and proteins participate in DNA replication
Enzymes in DNA replication Helicase unwinds Primase adds Binding proteins parental double helix short primer stabilize separate to template strand strands Ligase joins Okazaki DNA polymerase III DNA polymerase I fragments and seals binds nucleotides (Exonuclease) removes other nicks in sugar- to form new strands RNA primer and inserts phosphate backbone the correct bases
DNA Replication enzymes: DNA Polymerase • Catalyzes the elongation of DNA by adding nucleoside triphosphates to the 3’ end of the growing strand • A nucleoside triphosphate is a 1 sugar + 1 base + 3 phosphates • When a nucleoside triphosphate joins the DNA strand, two phosphates are removed. • DNA polymerase can only add nucleotides to 3’ end of growing strand
Complementary Base-Pairing in DNA • DNA is a double helix, made up of nucleotides, with a sugar-phosphate backbone on the outside of the helix. • Note: a nucleotide is a sugar + phosphate + nitrogenous base • The two strands of DNA are held together by pairs of nitrogenous bases that are attached to each other via hydrogen bonds. • The nitrogenous base adenine will only pair with thymine • The nitrogenous base guanine will only pair with cytosine • During replication, once the DNA strands are separated, DNA polymerase uses each strand as a template to synthesize new strands of DNA with the precise, complementary order of nucleotides.
DNA Replication enzymes: DNA Ligase • The two strands of DNA in a double helix are antiparallel (i.e. they are oriented in opposite directions with one strand oriented from 5’ to 3’ and the other strand oriented from 3’ to 5’ • 5’ and 3’ refer to the numbers assigned to the carbons in the 5 carbon sugar • Given the antiparallel nature of DNA and the fact that DNA ploymerases can only add nucleotides to the 3’ end, one strand (referred to as the leading strand ) of DNA is synthesized continuously and the other strand (referred to as the lagging strand ) is synthesized in fragments (called Okazaki fragments ) that are joined together by DNA ligase.
DNA Replication enzymes: Primase • DNA Polymerase can not initiate the synthesis of DNA • Remember that DNA polymerase can only add nucleotides to 3’ end of an already existing strand of DNA • Primase is the enzyme that can start an RNA chain from scratch and it creates a primer (a short stretch RNA with an available 3’ end) that DNA polymerase can add nucleotides to during replication. Note that the RNA primer is subsequently replaced with DNA
DNA Replication enzymes: Helicase, Topoisomerase and Single-strand binding protein • Helicase untwists the two parallel DNA strands • Topoisomerase relieves the stress of this twisting • Single-strand binding protein binds to and stabilizes the unpaired DNA strands
The starting material for PCR includes 1. Template DNA � Contains the region that needs to be amplified 2. Oligonucleotide primers � Complementary to sequences at the ends of the DNA fragment to be amplified. � Synthetic and about 20-30 nucleotides long 3. Deoxynucleoside triphosphates (dNTPs) � Provide the precursors for DNA synthesis 4. Taq polymerase � DNA polymerase isolated from the bacterium Thermus aquaticus This thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases
Rules for the design of PCR primers • Length of the primer: 20-30 bases • GC content: up to 50% • No complementarity between primers •Tm-value: 55-70 °C difference between both primers less than 2°C
The PCR process: 1. Initiation: one step for 2-5 min at 95°C 2. Denaturation 94 °C � separates both DNA strands from each others 3. Primer annealing ~50-60 °C � primer binds to complementary sequences on the target DNA. � Annealing temperature depends on the length and GC-content of the primer 4. Chain elongation 72 °C � The complementary strand is synthesized by the DNA polymerase
Standard thermocycle
Principle of Polymerase Chain Reaction = PCR A. Double 50º strand DNA B. 96º Denature 50º C. Anneal primers Taq D. 72º Polymerase binds Taq
1 After 5 rounds there are 32 double strands of 2 which 24 3 (75%) are of the same size 4 A typical PCR run is likely to involve 20 to 30 cycles of replication. • After 20 cycles, a DNA sample will increase 2 20 -fold (~ 1 million- fold). After 30 cycles, a DNA sample will increase 2 30 -fold (~ 1 billion-fold).
RT-PCR RT-PCR is the “reverse transcriptase- PCR”, which can be used to study gene expression. The steps: 1. mRNA isolation 2. Reverse transcription 3. PCR using specific primers. � The key enzyme is the reverse Transcriptase ( pol): � Reverse transcriptase is a RNA-depending DNA-Polymerase. � As DNA-Polymerase this enzyme needs a Primer Converts any RNA into a DNA • Pre-requisite: a complementary primer .
Agarose gel electrophoresis � separation range: 100bp to <50kb � separation accuracy: ~ 20 to 50bp
Parameters affecting migration of DNA through agarose gel � Agarose concentration � Electrophoresis buffer � DNA conformation � Applied voltage
Detection: fluorescence of ethidium-bromide under UV-light
Separation of DNA fragments in agarose gels Mixture of different DNA fragments long fragmen ts Plate of glass Short fragmen ts Finished DNA gel gel 21 electrophoresis
Applications of PCR � Diagnosis of genetic disorders � U sed to carry out a variety of tasks in molecular cloning and analysis of DNA. � Detection of nucleic acid sequences of pathogenic organisms in clinical samples. � Genetic identification of forensic samples (as in police laboratory) � Analysis of mutations in activated oncogenes
PCR has become a very powerful tool in molecular biology • One can start with a single sperm cell or stand of hair and amplify the DNA sufficiently to allow for DNA analysis and a distinctive band on an agarose gel. • One can amplify fragments of interest in an organism’s DNA by choosing the right primers. • One can use the selectivity of the primers to identify the likelihood of an individual carrying a particular allele of a gene.
PCR and Disease • Primers can be created that will only bind and amplify certain alleles of genes or mutations of genes • This is the basis of genetic counseling and PCR is used as part of the diagnostic tests for genetic diseases. • Some diseases that can be diagnosed with the help of PCR: • Huntington's disease • cystic fibrosis • Human immunodeficiency virus (HIV virus)
Huntington’s Disease (HD) • HD is a genetic disorder characterized by abnormal body movements and reduced mental abilities • HD is caused by a mutation in the Huntingtin ( HD ) gene • In individuals with HD, the HD gene is “expanded” – In non-HD individuals, the HD gene has a pattern called trinucleotide repeats with “CAG” occurring in repetition less than 30 times. – IN HD individuals, the “CAG” trinucleotide repeat occurs more that 36 times in the HD gene • PCR can be performed on an individual’s DNA to determine whether the individual has HD. – The DNA is amplified via PCR and sequenced (a technique by which the exact nucleotide sequence is determined) and the number of trinucleotide repeats is then counted.
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