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Polymerase Chain Reaction
Transcript of Polymerase Chain Reaction
Tumor cells displayed 3-fold lower random mutation frequency vs. normal colonic tissue Quantifying Random
Mutation Frequency Mitochondrial genome of all samples were sequenced by PCR amplification of mtDNA
Using 28 pairs of primers
Clonally expanded mutations scored only when adenoma or carcinoma mtDNA differed from normal colorectal tissue
Regions with mutations were reamplified and sequenced
Rule out possibility of mutations being produced by errors during PCR or sequencing mtDNA Genome Sequencing Methodology Measure frequency of non-clonal de novo mutations in mtDNA of normal and colorectal cancer tissues
Understand mitochondrial mutagenesis in normal and tumor cells
Identify possible mitochondrial targets for cancer prevention, treatment and diagnosis Purpose Background Human tumors often carry mutations in mtDNA
May drive cancer progression and metastasis
Assumption that mitochondrial genome in cancer is genetically unstable
Mutations in tumor cells provide genetic diversity
Fuels adaptive evolution and drives disease progression
Undergo positive selection and clonally proliferate Nolan G. Ericson, Mariola Kulawiec, Marc Vermulst, Kieran Sheahan, Jacintha O’Sullivan, Jesse J. Salk, Jason H. Bielas Decreased Mitochondrial DNA Mutagenesis in Human
Colorectal Cancer Begins at 3' end of primer making a double strand out of each single strands
Taq polymerase works in 5' -> 3' direction
dNTPs added to 3' end of primers to construct complementary strand of target sequence Extension Temperature is raised to approximately 72°C
Enzyme Taq DNA polymerase used to replicate DNA strands
Thermostable DNA polymerase from Thermus aquaticus
Begins synthesis at the region marked by primers Extension Heat (>90° C) separates double-stranded DNA into two single strands
Hydrogen bonds linking nucleotide bases are weak
Break at high temperatures
Covalent bonds between deoxyribose and phosphates remain intact Denaturation Basis of gene sequencing and RMC assay
Amplification of molecules containing mutations
Determination of mutation loci
Quantification of mutation frequencies in mitochondrial and nuclear DNA
Allow for comparison between cancerous and normal tissue genomes
Produce mtDNA copies for various biotechnological tests
Gas Chromatography Importance of PCR Hallmark mutator phenotype does not extend to mitochondria
mtDNA mutagenesis of single base substitutions is suppressed within human colorectal tumors
Cells with increased rate of nuclear mutagenesis may be selected for to facilitate generation with neoplastic advantage
Such pressures don't appear to exist for mitochondrial genome Genomic Instability Plotted random mutation frequency of all samples against the ratio of citrate to lactate
Examine relationship between energy metabolism and mtDNA mutation frequency
Mutation frequency naturally decreases with reduced mitochondrial respiration
Oxidative damage generated as a byproduct of OXPHOS causes mtDNA mutagenesis
Rationale for decreased frequency of random mutations in tumor cells Tumor cells reprogram energy metabolism from primarily oxidative phosphorylation (OXPHOS) to anaerobic glycolysis.
Called Warburg effect
Decreases OXPHOS and production of reactive oxygen species (ROS) in mitochondria
Analyzed relative expression of protein markers for glycolysis and OXPHOS (Fig. 4A)
Increase in glycolytic markers PK and GAPDH in tumors vs. normal tissue (Fig. 4B) Metabolic Shift C:G to T:A transitions predominant in normal & adenoma tissues (Fig. 3)
.3-fold decrease in C:G to T:A transitions in tumor samples vs. normal samples
Majority of mutations in carcinomas are C:G to T:A transitions
All tissue types showed similar levels of T:A to C:G mutations
Biological change responsible for reducing C:G to T:A mutations frequency occurs after initiation of neoplastic clonal expansion C∶:G to T∶:A Transitions Sequenced entire mitochondrial genome of each sample
55% (11 of 20) of carcinomas carried at least one clonally expanded mtDNA mutation
Protein-coding gene mutations resulted in frameshift (2/13) or non-synonymous changes (11/13)
Sequenced entire mitochondrial genome of 19 adenoma samples
To assess whether expansion of mtDNA mutations are early or late events during carcinogenesis
32% (6 of 19) of adenomas carried clonally expanded mutations
Clonal expansion of mtDNA mutations can occur prior to malignancy Stratifying Clonally Expanded Mutations GC is used to separate and analyzing compounds that can be vaporized without decomposition
Qualitate and quantitate different components of a mixture
MS measures the mass-to-charge ratio of charged particles
Measure the relative amount of citrate and lactate in colorectal tissues SDS-PAGE (separate proteins according to their size and no other physical feature)
Western Blot (determine molecular weight of a protein and measure relative amounts of protein present in different samples)
Western blot analysis of levels of OXPHOS markers and glycolytic pathways
Fractionated by SDS-PAGE and blotted with corresponding antibodies from normal and tumor tissues RMC-assay uses ability of TaqI (restriction enzyme) to discriminate between DNA molecules with wild type or mutant TaqI restriction site
After mtDNA digestion with TaqI, PCR is attempted across TaqI restriction site (red arrows)
PCR amplifies DNA molecules containing mutation in restriction site (red box)
Amplicons with wild type restriction site (blue box) are no longer PCR amplification templates
Second qPCR- quantifies every DNA molecule in a sample
Ratio of mutant molecules to total number of molecules is a direct measurement of mutation frequency mtDNA RMC Assay After a certain PCR product concentration, cycles no longer amplify exponentially
Depletion of "ingredients"
Amplified DNA from Real-Time PCR detected as the reaction progresses in real time
SYBR green only fluoresces when bound to double-stranded DNA Real-Time PCR Tumor tissue taken from 21 colorectal surgically treated cancer patients
2 Stage I, 11 Stage II, 7 Stage III & 1 Stage IV cancers
11/20 had matched adenoma tissue
8 had adenoma and matched normal tissue
Processed tissues centrifuged to isolate nuclei and cell debris
Supernatant centrifuged to pellet the mitochondria
mtDNA isolated by phenolchloroform extraction and isopropanol precipitation Tissue and DNA Isolation for Mutational Analysis Cycle is repeated ~40x
More than one billion exact copies of original DNA segment
Completed in a few hours
Directed by thermocycler
Alters temperature of reaction every few minutes Exponential Growth Two new DNA strands identical to original target sequence (amplicons)
DNA polymerase does not recognize end of sequence
Newly formed strands have a beginning
Defined by 5' end of the primer
3' end is not precisely defined Taq polymerase synthesizes new double stranded DNA molecules
Both identical to original double stranded target DNA sequence
Facilitates the binding and joining of dNTPs
Complementary nucleotides free in the solution Extension Takes place between 40° C and 65°C
Dependent on length and base sequence of the primers
Allows primers to anneal to target sequence with high specificity
Reaction buffer needed to facilitate primer annealing Annealing Primers to Target Sequences Goal of PCR is to replicate a unique target sequence of ~100-600 base pairs
One primer for each complementary single DNA strand
Start of target sequence marked by primers that anneal to complementary sequence Annealing Primers to Target Sequences A Recipe for PCR Technique used to amplify specific target sequences of DNA to permit further molecular and genetic analyses
Annealing Primers to Target Sequences
Extension Polymerase Chain Reaction (PCR) Four nitrogenous bases:
Purines (2 ringed base)
Adenine and Guanine
Pyrimidines (1 ringed base)
Cytosine and Thymine
Complementary nucleotides strands are linked during DNA replication DNA Hereditary material in most organisms
Composed of two complimentary nucleotide chains DNA (Deoxyribonucleic acid) Reduced mtDNA genetic diversity does not limit tumor progression
Normal mtDNA mutation rates may serve as barrier to cancer development
Mitochondrial theory of aging
ROS production, mtDNA damage, mutation, and respiratory chain dysfunction generate decline of mitochondrial function
Decrease in mtDNA mutagenesis may favor disease progression by breaking this cycle
Contributes to development of tumor cell immortality
Cancer therapy increasing mtDNA damage might suppress malignant growth Targeting mtDNA Mutagenesis Quantified frequency of random mutations in adenoma tissues
To assess whether clonal expansion alone accounts for decreased mutation load in tumor mtDNA
Presence of clonal mtDNA mutations in 32% of adenomas
Random mutation frequency in adenoma mtDNA higher than that of normal colorectal mtDNA (Figure 2C)
Figure 2D: Mean mtDNA random mutation frequency in carcinomas that harbored one or more clonal mutation Random Mitochondrial DNA Mutations mtDNA content per cell determined via real-time PCR in two separate reactions
With a primer set to mitochondrial DNA to quantify mitochondrial genomes
With a primer set to single copy nuclear ß-globin gene to quantify nuclear genomes mtDNA Copy Number
Quantification Mutation frequencies determined for TaqI digested mtDNA via real-time PCR in two separate reactions
With a primer set beside TaqI restriction site to quantify number of mutant molecules
With a primer set in a region without TaqI restriction site to quantify total number of molecules
PCR performed using Brilliant SYBR Green QPCR Master Mix , 25 forward and reverse primers, and diluted TaqI-digested mtDNA mtDNA RMC Assay Forensic
Extended Research Applications of PCR Carcinoma Tissues Normal Colorectal Tissues A)Mutation frequency at TaqI restriction sites 1215–1218 within the 12S rRNA gene
B)7335–7338 within the COXI gene
Mean mutation burden of carcinoma mtDNA is reduced ~3-fold vs. normal tissue Figure 1. Decreased Random Mitochondrial DNA Mutations in Colorectal Cancer Tumor tissue Normal tissue Abundance of OXPHOS marker, ß-F1-ATPase, shows decrease in mitochondrial bioenergetic competence in carcinomas vs to normal controls (Fig. 4C)
BEC index gives ratio of OXPHOS protein content to glycolytic protein content
Reduced BEC in tumor tissue (Fig. 4D)
GC/MS metabolite analysis shows higher levels of lactate (glycolysis) and lower levels of citrate (OXPHOS) in cancer tissues vs normal tissues (Fig. 4E)
Consistent with Warburg effect Metabolic Shift http://www.scfbio-iitd.res.in/tutorial/gene.html Works Cited
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