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Polymerase Chain Reaction (PCR) (Definition)

-in vitro process modeled after cellular DNA replication, was developed by Kary Mullis as a method for amplifying very small quantities of DNA
-major advance in field of DNA clongin and analysis


PCR (Sequence)

-first two DNA strands are separated called denaturing step
-next primers are glued to DNA strand called annealing step
-third involve allowing DNA plymerase to extend the primer sequence by synthesizing new strands of DNA
-all three together is a cycle
-DNA doubles after each cycle


PCR (Ingredients)

-cellular respiration usually requires many proteins, PCR designed to use only one: DNA polymerase
-single PCR reaction consists of DNA template, set of oligonucleotide primers, DNA polymerase and a solution containing the four DNA nucleotides and a physiologically relevant salt mixture


PCR (Derivation of Ingredients)

-template is usually derived from either genomic or plasmid DNA and is usually maintained at very low concentrations within the reaction mixture
-two oligonucleotide primers are designed by the researcher to bind to sites within the template, are synthesized by machines and then added to the reaction mixture
-primers are designed to anneal to the ends of the DNA sequence that you want to amplify
-one primer will complement one of the two strands while the other will complement the other strand
-DNA polymerase that is added to the mixture has been genetically/molecularly modified to withstand high reaction temperatures, to have a high degree of fidelity and to be processive in the absence of the sliding clamp
-reaction mixture contains salts, metal ions and nucleotides
-nucleotides are of course used by the polymerase in the synthesis of new DNA strands while the salts and metals are present to mimic the nuclear environment in which the polymerase functions best


Cellular DNA Replicaton vs PCR

-denaturing achieved by heating the reaction to approximately 95°C which breaks hydrogen bonds that exists between nucleotides thereby allowing for the separation of the two polynucleotide strands
-differs from cellular DNA replication in which double helix unwound by DNA helicase
-consequence of heat heat to completely melt the the two strands apart replication forks do not form and positive supercoiling does not take place so no requirement for Topoisomerase II in reaction mixture


Second Step of PCR in depth

-(annealing) involves cooling of the reaction to approximately 68°C allows for short oligonucleotide primers to base-pair with complementary sequences within the template (~30 seconds) allows for annealing of the primers to the template without having the two template strands coming back together


Third Step of PCR in depth

-(elongation) involves the synthesis of new strands by DNA polymerase. The temperature of the reaction is elevated to ~72°C since that is the optimal temperature for most modified polymerases
-since replication forks are not made both strands are synthesized in continuous stretches (no leading or lagging strands)
-primer sequences are not removed therefore a PCR reaction also does not require RNase, the exonuclease or DNA ligase


Gel Electrophoresis (What)

-technique that has been developed to view and analyze DNA fragments
-fragments can be derived from PCR (previous slides) or from the digestion of DNA using restriction enzymes
-uses an electrical charge to separate DNA fragments by their relative size


Prior to Gel Electrophoresis

-a powder of agarose is mixed with a salt buffer containing ethidium bromide, boiled in a microwave, poured into a mold and allowed to cool
-final gel is a porous matrix and has a consistency that is similar to Jell-O
-agarose gel slab is then placed into a box that is filled with a salt buffer and ethidium bromide


Gel Electrophoresis (Process)

-solution of DNA is then mixed with a heavy dye (which interacts with nucleic acids and makes it heavier) and loaded into precut wells of the gel
-this mixture sinks to bottom of well
-electric current is applied to the gel
-negative electrode (cathode) is located near the wells and the positive electrode (anode) is placed away from the wells
-DNA is negatively charged the individual fragments will migrate through the gel towards the positive electrode
-DNA fragments are of different sizes, the smaller pieces can migrate faster than the larger pieces
-The DNA fragments can be viewed by placing the gel on an ultraviolet light emitting box
-ethidium bromide molecules absorb the UV light and emit light in the orange-red spectrum


Restriction Enzymes

-proteins that digest (cut) DNA at specific sequences called restriction sites
-found in bacteria as well as archaea and are thought to be part of a defense system against viral and phage infections
-several thousand have been identified
-each can recognize a unique DNA sequence (ranging from 4-12 nucleotides in length)
-typically will bind to DNA as homodimer
-once bound to correct sequence of nucleotides makes a double-stranded break
-in general these enzymes will digest unmethylated DNA
-Bacterial/archaea DNA is heavily methylated (by enzymes called methylases) and is therefore protected from the activity of restriction enzymes
-phage and viral DNA are unmethylated thereby making perfect targets for restriction enzymes


Restriction Enzymes in Research Labs

-have been heavily used in DNA cloning and in diagnostic tests
-in gel electrophoresis each lane of the agarose gel contains many DNA fragments
-fragments were initially part of a larger piece of DNA that was digested with restriction enzymes
-pattern of restriction digest fragments can be used to make crude judgments on the degree of similarity between pieces of DNA


Replication Enzymes Recognition Sequences

-these enzymes recognize specific nucleotide sequences and can range from 4-12bp
-inverse relationship between the size of the recognition sequence and the frequency at which it is found in the genome


Different Ends of Fragments

-fragments that result form restriction enzyme activity have one of three different types of ends
-some enzymes leave blunt end which means that that the double stranded breaks occurred at the same position on each strand
-other enzymes will make asymmetric cuts and will leave fragments containing either 5` or 3` end overhangs called sticky or compatible end


Products of Restriction Digests

-can be glued back together again using DNA ligase
-however (1) sticky ends must be compatible (digested with same enzyme) in order for them to be glued together
-(2) frangmets with blunt ends can be glued together irrespective of the restriction enzyme used to generate the end
-(3) fragment with blunt ends cannot be glued to a fragment with sticky ends


Determining Sequence of DNA

-methods to do this pioneered by Fred Sanger and Walter Gilbert
-over decades sequencing methods have become fast and inexpensive and entire genomes can be sequenced in same time that it used to take to sequence 100 bases of DNA


Modern Method of DNA Sequencing

-strands of a DNA molecule are separated with sodium hydroxide and affixed to a glass slide
-short primer sequence is added and allowed to pair to complementary sequences within the template
-modified versions of all four bases are added to the reaction
-each base is modified so that new bases cannot be added after one has been incorporated and each type of base has been labeled with a different colored dye
-once base is added all other bases are washed away and the color of the base is recorded using a color detector
-a laser beam is focused on the DNA strand which removes the colored dye and the blocking modification
-new nucleotides are added and the process is repeated
-in this way, entire sequence of a DNA fragment can be determined


PCR and DNA Sequencing in Everyday

-over last two decades PCR has been used to amplify extremely small amounts of DNA thus allowing for scientists to provide answers to questions that until recently remained unanswered
-PCR followed by DNA sequencing has been used successfully within the field of forensic science to (1) provide identities to human tissues and remains and (2) to convict or exonerate individuals who have been accused of committing crimes
-. DNA can be isolated and amplified from a small drop of blood or a single hair cell or skin cell
-PCR has also been used to amplify small quantities of DNA that is present within the remains of extinct organisms such as old hominoids like Homo erectus and Neanderthal and other extinct organisms such as woolly mammoths
-PCR can also be used to answer historical questions. For example, at one point there were doubts if the man buried in Jesse James’ tomb is in fact the legendary bank robber
-or some have thought that President Lincoln had Marfan’s syndrom
-or discussions of President Jefferson fathering a child that was born of a slave
-PCR can also be used in medical diagnostic tests to detect early stages of cancers or other diseases. At these early stages the afflicted tissues may be no more than a few abnormal looking cells