DNA mutations

http://www.ncbi.nlm.nih.gov/books/NBK21897/

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Types of mutations

• Micromutation that involve small regions of the DNA

• Macromutations that involve the chromosomes as a whole

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DNA micromutations

• Single-base mutations can result in different types of mutations such as:

–missense: a change of an amino acid

–nonsense: premature termination of protein synthesis

–frameshift: altering the reading of amino acid sequence

–silent: does not lead to any change at the protein level, but contributes to genetic variability among individuals

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DNA micromutations (cont.)

• Translocations, that bring different regions of gene segments together,

• Deletions of a few nucleotides to long stretches of DNA,

• Insertions and duplications of nucleotides or long stretches of DNA, and

• Inversion of DNA segments

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Causes of DNA mutation

• DNA mutations can arise spontaneously or induced

• Spontaneous mutations are naturally occurring mutations and arise in all cells

• Induced mutations are produced when an organism is exposed to a mutagenic agent, or mutagen

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Spontaneous mutations

• Spontaneous mutations arise from a variety of sources, including errors in DNA replication and spontaneous lesions

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Errors in DNA replication

• An error in DNA replication can occur when an inaccurate nucleotide pair (say, A-C instead of G-C) forms in DNA synthesis, leading to a base substitution

• Each of the bases in DNA can appear in one of several forms, called tautomers

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Effect of tautomers

• Following DNA replication, tautomers lead to either transition or transversion mutations

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Transitions

• A purine substitutes for a purine or a pyrimidine for a pyrimidine

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Transversions

• In transversion mutations, a pyrimidine substitutes for a purine or vice versa

• In this case, a replication error would require mispairing of a purine with a purine or a pyrimidine with a pyrimidine

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Other errors of DNA replication

• Frameshift mutations

• Deletions and duplication

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Frameshift mutations

• These mutations often occurred at repeated sequences

• Such mutations change the reading "frame" of codons and leads to changes in the amino acid sequence of the produced protein

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Deletions and duplications

• Large deletions (more than a few base pairs) often occur at repeated sequences

• Duplications of segments of DNA have also been observed

• Like deletions, they often occur at sequence repeats

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Spontaneous lesions

• Spontaneous lesions are naturally occurring type of DNA damage that can generate mutations

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Types of spontaneous lesions

• Depurination

• Deamination

• Oxidatively damaged bases

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Depurination

• Cleavage of the glycosidic bond between the base and deoxyribose

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Deamination

• The deamination of cytosine yields uracil

• Uracil residues will pair with adenine during replication, resulting in the conversion of a G-C pair into an A-T pair (a GC→AT transition)

Methylated cytosine

• Methylated cytosine can also be methylated resulting in its conversion to thymine and leading to a transition mutation

Oxidatively damaged bases

• Active oxygen species such as hydrogen peroxide (H2O2) are normally produced during metabolism

• They can damage DNA, which results in mutation

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8-oxodG

• A product of oxidative damage is 8-oxo-7-hydrodeoxyguanosine (8-oxodG, or GO) product frequently mispairs with A, resulting in a high level of G→T transversions

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INDUCED MUTATIONS

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Mechanisms of mutagenesis

• Mutagens induce mutations by at least three different mechanisms:

– replace a base in the DNA

– alter a base so that it specifically mispairs with another base

– damage a base so that it can no longer pair with any base under normal conditions

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Incorporation of base analogs

• Some chemical compounds are similar to the normal bases of DNA that they are incorporated into DNA in place of normal bases; such compounds are called base analogs

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Mechanism of mutation

• These analogs can produce mutations by causing incorrect nucleotides to be inserted opposite to them during replication

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5-bromouracil

• 5-bromouracil (5-BU) is an analog of thymine

• The normal structure of 5-BU pairs with adenine

• When ionized, 5-BU pairs with guanine

• 5-BU causes transition mutations

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Specific mispairing

• Some mutagens are not incorporated into the DNA but can cause specific mispairing

– For example, the addition of an alkyl group to guanine leads to the formation of O-6-alkylguanine and results in direct mispairing with thymine, and consequently a GC AT transitions

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Intercalating agents

• The intercalating agents such as proflavin and ethidium bromide are planar molecules that can insert themselves (intercalate) between the stacked nitrogen bases imitating base pairs

• The intercalated agent can cause single-nucleotide-pair insertions or deletions

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Base damage

• A large number of mutagens damage one or more bases, so no specific base pairing is possible

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Ionizing radiation

• Ionizing radiation results in the formation of ionized and excited molecules that can cause damage to cellular components including DNA

• Many different types of reactive oxygen species are produced that can

– damage bases,

– cause breakage of the N-glycosidic bond (AP sites), or

– cause strand breaks

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Mutagens and carcinogens

• Mutagenicity and carcinogenicity are clearly correlated when most mutagens are also carcinogens (approximately 90 percent)

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Tests of mutagenicity

• Rapid tests make use of microbes to test for mutagenicity

• The most widely used test was developed in the 1970s by Bruce Ames, who worked with Salmonella typhimurium

http://www.ncbi.nlm.nih.gov/books/NBK21788/33

Features of Salmonella typhimurium

• The Ames test uses a mutant strain of S. typhimurium

– cannot grow in the absence of the amino acid histidinebecause a mutation has occurred in a gene that encodes one of the enzymes necessary for histidine biosynthesis

– They also carry a mutation that inactivates a DNA repair system

– They also carry a mutation that eliminates the protective lipopolysaccharide coating of wild-type Salmonella to enable the entry of many different chemicals into the cell

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Reversion mutation

• In order for these cells to survive in the absence of histidine, they must have a mutation that corrects the original mutation that prevented the production of the missing enzyme

• This type of mutation is known as reversion, because this second mutation returns the mutant to the wild-type genotype

• This reversion can happen spontaneously or as the result of a mutagen

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What is considered a mutagen?

• A compound must increase the number of colonies grown in the absence of histidine by doublecompared to cells grown in the absence of the compound

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Example

Water Motor oil Alcohol Drug X قروش 5شيبس أبو

10 50 43 9 200

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Role of liver enzymes

• In mammals, chemicals are normally detoxified or broken down by liver enzymes

• In some cases, the action of liver enzymes can create a toxic or mutagenic compound from a substance that was originally safe

• Ames incorporated mammalian liver enzymes in his bacterial test system

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Example

Condition Water Motor oil Alcohol Drug X قروش 5شيبس أبو

-liver enzymes 10 50 43 9 200

+liver enzymes 12 22 50 35 500

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Significance of Ames test

• Chemicals detected by this test can be regarded as potential carcinogens

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DNA repair mechanisms

Resources

• This lecture

• Campbell and Farrell’s Biochemistry, pp. 263-268

• An Introduction to Genetic Analysis. 7th edition. Griffiths AJF, Miller JH, Suzuki DT, et al., New York: W. H. Freeman; 2000. http://www.ncbi.nlm.nih.gov/books/NBK22004/

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DNA repair

• Maintaining genetic stability requires not only an accurate mechanism of DNA replication, but also mechanisms for repairing DNA damage

• These mechanisms are collectively called DNA repair

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Repair mechanisms

• Prevention of errors before they happen

• Direct reversal of damage

• Excision-repair pathways

• Postreplication repair

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PREVENTION OF ERRORS BEFORE

THEY HAPPEN

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Reactive oxygen species

• Some enzymatic systems neutralize potentially damaging compounds before they even react with DNA

• One example of such a system is the detoxification of reactive oxygen species and oxygen radicals, which are produced during oxidative damage to DNA

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Superoxide dismutase

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Catalase

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DIRECT REVERSAL OF DAMAGE

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Cyclobutane pyrimidine

• Some lesions can be repaired by reversal of DNA damage

• UV light that hits DNA results in the formation of a covalent interaction between two adjacent pyrimidine bases forming structures known as cyclobutane pyrimidinedimers, most frequently between two thymines

• This product is a mutagenic photodimer

Photolyase

• Cyclobutane pyrimidine can be repaired by a photolyase that has been found in bacteria and lower eukaryotes but not in humans

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8-oxodG

• The enzyme product of the mutT gene prevents the incorporation of 8-oxydeoxyguanosine (8-oxodG) into DNA

• 8-oxodG is formed from free radical attack of DNA and pairs with A rather than C

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EXCISION-REPAIR PATHWAYS

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Types

• General excision repair

• Coupling of transcription and repair

• Specific excision pathways

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General excision repair

• Also termed nucleotide excision repair

• This system includes the breaking of a phosphodiester bond on either side of the lesion, on the same strand, resulting in the excision of an oligonucleotide

• This excision leaves a gap that is filled by repair synthesis, and a ligase seals the breaks

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The mechanism

• In E. coli, the UvrA protein recognizes the damaged DNA, forms a complex with UvrB and leads the UvrB subunit to the damage site

• The UvrC protein then binds to UvrB

• Each of these subunits makes an incision (a cut)

• The short DNA is unwound and released by a helicase

• DNA polymerase I then fills in the gap, followed by the action of DNA ligase

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In human…

• In human cells, the process is more complex than its bacterial counterpart

• However, the basic steps are the same as those in E. coli

• Defect in this mechanism causes a condition known as Xeroderma pigmentosum

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Transcription and repair

• In both eukaryotes and prokaryotes, there is a preferential repair of the transcribed strand of DNA for actively expressed genes

• RNA polymerase pauses when encountering a lesion

• The general transcription factor TFIIH and other factors carry out the incision, excision, and repair reactions

• Then transcription can continue normally

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SPECIFIC EXCISION PATHWAYS

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DNA glycosylase repair pathway

• DNA glycosylases do not cleave phosphodiester bonds, but instead cleave N-glycosidic (base-sugar) bonds of damaged bases , liberating the altered base and generating an apurinicor an apyrimidinic site, both called AP sites

• The resulting AP site is then repaired by an AP endonucleaserepair pathway

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DNA glycosylases

• Numerous DNA glycosylases exist

• One, uracil-DNA glycosylase, removes uracil from DNA

• Uracil residues, which result from the spontaneous deamination of cytosine can lead to a CT transition if unrepaired

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AP endonuclease repair pathway

• The AP endonucleases introduce chain breaks by cleaving the phosphodiester bonds at AP sites

• This bond cleavage initiates an excision-repair process mediated by an exonuclease, DNA polymerase I, and DNA ligase

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GO system

• mutM removes 8-oxodG, or GO, lesion from DNA

• If DNA is replicated,

– mutM first removes the GO lesion

– Then, mutY removes the mispairedadenine from the opposite strand, leading to restoration of the correct cytosine by repair synthesis (mediated by DNA polymerase I) and allowing subsequent removal of the GO lesion by the mutM product

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POSTREPLICATION REPAIR

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Mismatch repair

• Some repair pathways are capable of recognizing errors even after DNA replication has already occurred

• One such system, termed the mismatch repair system, can detect mismatches that occur in DNA replication

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The mechanism

• Recognize mismatched base pairs.

• Determine which base in the mismatch is the incorrect one.

• Excise the incorrect base and carry out repair synthesis

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The mechanism in details

• MutS binds to mispair.

• MutH and MutL are recruited to form a complex

• MutH cuts the newly synthesized (unmethylated) strand, and exonucleasedegradation goes past the point of the mismatch, leaving a patch

• Single-strand-binding protein (Ssb) protects the single-stranded region across from the missing patch

• Repair synthesis and ligation fill in the gap

MutS

MutL

MutH

Ssb

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But….

• In a G-T mismatch, both are normal bases in DNA

• How can the mismatch repair system determine whether G or T is incorrect?

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DNA methylation

• DNA is methylated following replication by the enzyme, adenine methylase

• However, it takes the adenine methylase several minutes to methylate the newly synthesized DNA

• The mismatch repair system in bacteria takes advantage of this delay to repair mismatches in the newly synthesized strand

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In humans

• The mismatch repair system has also been characterized in humans

• Two of the proteins, hMSH2 and hMLH1, are very similar to their bacterial counterparts, MutS and MutL, respectively

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SOS system

• A large number of mutagens such as ultraviolet light damage one or more bases, resulting in a replication block, because DNA synthesis will not proceed past a base that cannot specify its complementary partner by hydrogen bonding

• In bacterial cells, such replication blocks can be bypassed by inserting nonspecific bases. The process requires the activation of a special system, the SOS system

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Proteins of SOS

• Exactly how the SOS bypass system functions is not clear, although in E. coli it is known to be dependent on at least three genes, recA, umuC, and umuD

• Current models for SOS bypass suggest that the UmuC and UmuD proteins combine with the polymerase III DNA replication complex

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The mechanism

• DNA polymerase III pauses at a type of damage called a TC photodimer

• This region attracts single-strand-binding protein (Ssb), as well as the RecA protein, which signals the cell to synthesize the UmuC and UmuD proteins forming the SOS system

• The SOS system allows DNA polymerization to continue past the dimer

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SOS system and mutations

• However, the SOS system inserts the necessary number of bases (often incorrect ones) directly across from the lesion and replication continues without a gap

• SOS system often generates mutations

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Recombinational repair

• The recA gene also takes part in postreplication repair

• Here the DNA replication system pauses at a UV photodimeror other blocking lesion and then restarts past the block, leaving a single-stranded gap

• In recombinational repair, this gap is patched by DNA cut from the sister molecule

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MUTATORS

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What is a mutator?

• Mutant strains with increased spontaneous mutation rates have been detected

• Such strains are termed mutators

• In many cases, the mutator phenotype is due to a defective repair system

• In humans, these repair defects often lead to serious diseases

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Disease related to DNA repair

Condition Sensitivity Cancer susceptibility

Mutated gene

Ataxia telangiectasia

γ irradiation Lymphomas ATM (cell cycle and DNA repair)

Bloom syndrome Mild alkylating agents

Carcinomas, leukemias, lymphomas

ATM (cell cycle and DNA repair)

Cockayne syndrome UV light ERCC6 or ERCC8(DNA repair)

Fanconi anemia Cross-linking agents Leukemias Multiple (response to DNA damage)

Xerodermapigmentosum

UV light, chemical mutagens

Skin carcinomas and melanomas

Nucleotide excision repair

HNPCC Colon, ovary Mismatch repair79

Hereditary nonpolyposis colon

cancer (HNPCC)

• Several genes responsible a type of colon cancer have been identified, including MSH2 and MLH1

• The protein products of the MSH2 and MLH1 genes appear to have critical roles in the recognition and repair of DNA mismatches

• They are homologous to the mutL and mutS genes in bacteria and yeast

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DNA sequencing and PCR

Resources

• This lecture

• Campbell and Farrell’s Biochemistry, pp. 377-380, 384-387

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What is DNA sequencing?

• DNA sequencing is the process of determining the exact order of the chemical building blocks, that are the A, T, C, and G bases, that make up the genome

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Importance of knowing the DNA

sequence

• Identification of genes and their localization

• Identification of protein structure and function

• Identification of DNA mutations

• Genetic variations among individuals in health and disease

• Prediction of disease-susceptibility and treatment efficiency

• Evolutionary conservation among organisms

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DNA sequencing of organism

genome

• Viruses and prokaryotes first

• This was followed by the determination of the sequence of human mitochondrial DNA

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Then simple eukaryotes

• The first eukaryotic genome to be completely sequenced was that of yeast, Saccharomyces cerevisiae, which comprises approximately 12 million base pairs, distributed on 16 chromosomes

• This achievement was followed by the first complete sequencing of the genome of a multicellular organism, the nematode Caenorhabditis elegans, which contains nearly 100 million base pairs

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Last, but not least…

• In humans, it is determining the sequence of the 3 billion bases

• Determination of the base sequence in the human genome was initiated in 1990 and completed in May 2006 via the Human Genome Project

• Achieving this goal has helped reveal the estimated 20,000-30,000 human genes within our DNA as well as the regions controlling them

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Method of DNA sequencing

• Based on premature termination of DNA synthesis by dideoxynucleotides

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The process…

• DNA synthesis is initiated from a primer that has been labeled with a radioisotope

• Four separate reactions are run, each including deoxynucleotides plus one dideoxynucleotide (either A, C, G, or T)

• Incorporation of a dideoxynucleotide stops further DNA synthesis because no 3 hydroxyl group is available for addition of the next nucleotide

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Generation of fragments

• A series of labeled DNA molecules are generated, each terminated by the dideoxynucleotide in each reaction

• These fragments of DNA are then separated according to size by gel electrophoresis and detected by exposure of the gel to X-ray film

• The size of each fragment is determined by its terminal dideoxynucleotide, so the DNA sequence corresponds to the order of fragments read from the gel

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Fluorescence-based DNA

sequencing

• Large-scale DNA sequencing is frequently performed using automated systems using fluorescence-based reactions using labeled ddNTPs

• In this case, all four terminators can then be placed in a single tube, and only one reaction is necessary

• The reactions are run into one lane on a gel and a machine is used to scan the lane with a laser

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Detection of fragments

• The wavelength of fluorescence can be interpreted by the machine as an indication of which reaction (ddG, ddA, ddT, or ddC) the product came from

• The fluorescence output is stored in the form of chromatograms

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Why is fluorescence better than

radioactivity?

• It eliminates the use of radioactive reagents

• Can be readily automated

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Polymerase Chain Reaction

• In 1984, Kary Mullis devised a method called the polymerase chain reaction (PCR) for amplifying specific DNA sequences

• PCR allows the DNA from a selected region of a genome to be amplified a billionfold, effectively "purifying" this DNA away from the remainder of the genome

• The PCR method is extremely sensitive; it can detect a single DNA molecule in a sample

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Components of PCR reaction

• A pair of primers that hybridize to the target DNA.

– These primers should be specific for the target sequence and which are often about 15-25 nucleotides long. The region between the primers is amplified

• All four deoxyribonucleoside triphosphates (dNTPs: dATP, dCTP, dGTP and dTT)

• A heat-stable DNA polymerase

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DNA polymerases

• Suitably heat-stable DNA polymerases have been obtained from microorganisms whose natural habitat is hot springs

• For example, the widely used Taq DNA polymerase is obtained from a thermophilic bacterium, Thermus aquaticus, and is thermostable up to 94°C

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PCR cycle

• Denaturation, typically at about 93-95°C. At this temperature the hydrogen bonds that hold together the two polynucleotides of the double helix are broken, so the target DNA becomes denatured into single-stranded molecules

• Reannealing at temperatures usually from about 50°C to 70°C where the primers anneal to the DNA

• DNA synthesis, typically at about 70-75°C, the optimum for Taq polymerase

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PCR cycles

• 20-30 cycles of reaction are required for DNA amplification,

– the products of each cycle serving as the DNA templates for the next-hence the term polymerase "chain reaction“

• Every cycle doubles the amount of DNA

• After 30 cycles, there will be over 250 million short products derived from each starting molecule

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Detection of DNA fragments

• This DNA fragment can be easily visualized as a discrete band of a specific size by agarose gel electrophoresis

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Importance of primers

• The specificity of amplification depends on the specificity of the primers to not recognize and bind to sequences other than the intended target DNA sequences

• How can you prevent it?

• How can you take advantage of it?

Annealing temperature

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Disadvantage of PCR

• Primers must be known

• Contamination

• Product length is limited (usually <5 Kb)

• Accuracy is an issue

• Not quantitative

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What are the strengths of PCR?

• Easy, fast, sensitive, robust

• Discovery of gene families

• Disease diagnosis

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Forensic medicine

• An individual DNA profile is highly distinctive because many genetic loci are highly variable within a population

• PCR amplification of multiple genes is being used to establish paternity and criminal cases

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Quantitative PCR (qPCR)

• Measurement of amount of DNA in a sample

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•http://www.youtube.com/watch?v=kvQWKcMdyS4•http://www.bio.davidson.edu/courses/immunology/flash/rt_pcr.html

Components of the human genome

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Repetitive DNA sequences

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Tandem vs. dispersed

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Satellite (macro-satellite) DNA

• Repeats of 100 to 6500 bp

• Tandemly centromeric repeats (171 bp) unique to each chromosome

» Each chromosome has its unique sequence

» possible to make DNA probes specific to each

• Telomeric repeats

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VNTRs (minisatellite)

• Mini satellite sequences or VNTRs (variable number of tandem repeats)

• They are composed of 20 to 100 bp repeats

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STRs (microsatellites)

• STRs (short tandem repeats) composed of 2 to 10 bp repeats

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Polymorphisms of VNTR and STR

• The number of repeats in micro and mini satellites are highly variable (polymorphic)

• Useful in:

• Gene mapping

• DNA profiling for paternity testing, forensic testing, confirmation of relatedness and dead body identification

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The bad side of STRs

• Reduction or expansion of STR can be pathogenic

• Unstable expansion of short tandem repeats is characterised by anticipation

• Disease associated with large expansions outside coding sequences

• Myotonic dystrophy (DM1)

• Friedrich ataxia (FA)

• Disease associated with modestexpansions outside coding sequences– Huntington disease (HD)

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PCR of VNTRs

• VNTR blocks can be extracted and analyzed by RFLP or PCR and size determined by electrophoresis

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VNTR in medicine and more

• The picture below illustrates VNTR allelic length variation among 6 individuals.

The likelihood of 2 unrelated individuals having same allelic pattern extremely improbable

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SINEs and LINEs

• Two thirds (66.7%) of the repetitive non-coding DNA sequences

• Dispersed throughout the genome

• Divided into short and long interspersed sequences, SINES and LINES

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LINEs (Long interspersed elements)

• 7000 bp in length

• Represent about 4% of our total human genome

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SINEs (short interspersed elements)

• Shorter interspersed elements 90 to 500 bp in length

– Example: Alu sequence (~300 bp)

• Alu sequences are unique to humans (and some apes)

• 10% of the total human genome

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Transposons

• SINEs and LINEs are transposable (jumping or moving) elements

• Their origin is retroviruses

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Effect of trasnposons

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Pathogenic transpososns

• Transposable elements can cause mutations (Hemophilia) and gene rearrangements and duplication (e.g., beta globin genes)

• Transposons are mutagens

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Single nucleotide polymorphism (SNPs)

• Another source of genetic variation

• Single-nucleotide substitutions of one base for another

• Two or more versions of a sequence must each be present in at least one percent of the general population

• SNPs occur throughout the human genome - about one in every 300 nucleotide base pairs.

– ~10 million SNPs within the 3-billion-nucleotide human genome

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Categories of SNPs

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