Molecular Biochem Quiz 3

Reverse transcriptase (hybridization of oligonucleotide-T7-promoter primer to target sequence)

(generation of RNA:DNA hybrid) Single stranded RNA added and primer 1 added AMV-RT (reverse transcriptase) will generate cDNA from an RNA template (reverse transcription)

result after 30 cycles

1 x 10^9

amplification product longer targets

100-1500 bp 25 kb

CDC use of PCR

1995- CDC implemented use of rDNA PCR for population-based surveillance of unexplained life-threatening infections in the Unexplained Deaths and Critical Illnesses project Rapidly became gold standard to ID bacteria Metabolic genes can also be amplified to determine ID of bacteria Methicillin resistance of spathylococcus aureus is mediated by mecA gene which encodes penicillin-binding protein 2a Presence of mecA gene identifiable by PCR Goal of this unit: Use PCR to amplify 16S rRNA gene using universal primers and the metabolic gene (lacZ) using a specific set of primer pairs from a pure bacterial culture E. coli is bacterial source here Contains three structural genes termed the lac operon needed for transport and metabolism of lactose lacZ gene encodes beta-galactosidase

Normally, ____ cycles performed in each PCR run (one round of all three steps) and amount of DNA doubles after each cycle

30-40

Explain real-time PCR

Amount of fluorescence proportional to amount of PCR product in the reaction after each cycle Standard curve based on known amounts of target DNA, we can quantitate amount of target DNA in a test sample PCR amplification/detection takes place in a tube, much faster than end-point analysis (Yes or No answers immediately) Two strategies: TaqMan oligonucleotide strategy (Applied Biosystems) or SYBR green binding to synthesized double-strand DNA Allows accurate and precise quantification of product during the exponential phase, where amplification rate is similar across samples regardless of target amount Results are more sensitive than conventional PCR, where products measured during plateau phase and differences in target levels not proportional Probes are oligonucleotides labeled with fluorescent reporter and Quencher Allows us to know initial concentration Fluorescence reporter released from probe for every amplification Reporter molecule only released when a DNA strand completely polymerized by TAQ Optical module records fluorescence intensity Converted into amplification plot/curve

Enzymes in reaction

Based on simultaneous activity of avian myeloblastosis virus reverse transcriptase (AMV RT), RNase H, and T7 RNA polymerase with two oligonucleotide primers to produce amplification of desired fragment by more than 10^12 fold in 90-120 minutes All enzymatic reactions take place concurrently and reduce time for procedure Nucleic acids are a template for the amplification reaction only if single stranded and located in the primer-binding region Isothermal (one temperature of 41 degrees Celsius, no need for thermocycler) This means amplification of ssRNA possible if denaturation of dsRNA is prevented in the sample preparation procedure Possible to pick up mRNA in a dsDNA background without getting false positive results caused by genomic dsDNA in contrast with RT-PCR (would pick up dsDNA)

Describe with a diagram, how a single target is amplified in the branched DNA signal amplification technique.

Branched DNA assay is a signal amplification (as opposed to a target amplification) that is used to detect nucleic acid molecules Detection sensitivity provided by amplification of signal from probe Uses "capture probes," "bDNA probes," and "bDNA amplifier probes" Based upon microtiter plate technology From the base up, a branched DNA assay begins with a dish or some other solid support like a dipstick Signal amplification on the bDNA probe after direct binding of a large hybridization complex to the target sequence "Sandwich" complex of probes and target sequence One end of bDNA binds a specific target and other end contains branches that are designed to bind to a probe for signal detection These oligonucleotides are composed of a primary sequence and secondary sequences that result in a branched structure extending from primary sequence

State the enzymatic method one can use to prevent contamination due to previous amplified PCR products

Contamination control Pre and post-PCR Negative template control reaction with reagent check (10x buffer, dNTP, primers) Use of barrier pipet tips, pipet-positive displacement tips PCR reaction- substitute dTTP with dUTP Amplicons (PCR products U instead of T) New PCR reaction- add Uracil-N-Glycoslylase and UNG destroys any carryover

Describe components involved in PCR

DNA polymerase (synthesizes DNA from deoxyribonucleotides)-- usually Taq Four deoxynucleotides (dNTPS- dATP, dGTP, dCTP, dTTP)- building blocks of DNA replication, losing two phosphate groups in the process of incorporation Primers for both DNA strands (sense and anti-sense)- sense is the coding strand or non-template strand, antisense is non-coding strand and is the template for synthesis of RNA DNA primer is a short strand of RNA/DNA that serves as a starting point for DNA synthesis and is required for DNA replication because polymerase can only add new nucleotides to an existing strand of DNA Template dsDNA Appropriate buffer system (Tris, ammonium ions and/or potassium ions, magnesium ions)

Cocktail of what can be used to rescue target DNA for PCR application

DNA repair enzymes

Identify some of diagnostic uses for TAS and branched DNA amplification technique.

Detection of HIV and other viruses (hepatitis, HTLV, CMV)

Describe denaturation, annealing, and polymerization steps in a PCR cycle

Each amplification cycle of PCR involves three steps: denaturation, annealing, elongation Denaturation- mixture is heated to 95 degrees C to separate or "melt" the dsDNA template Annealing- performed at a lower temperature (usually 50-60 degrees Celsius and determined by the melting temperature of the primer, or Tm) and allows the primer to bind to the complementary sequence on a strand of the DNA template Recombination of DNA in the double stranded form following separation by heat Polymerization/elongation- DNA polymerase extends DNA synthesis from the primer and proceeds at 68-72 degrees Celsius Allows special polymerase that functions at higher temperature, like Taq polymerase from Thermus aquaticus to rapidly copy the DNA

describe DNA polymerase in PCR

Enzyme originally isolated from a thermophilic (heat-loving) bacterium called Thermus aquaticus found in hot springs and functionally stable at very high temperatures like PCR denaturation at 94 degrees Celsius Only needs to be added once at the beginning of the PCR run (will not be denatured)

Why is contamination a major concern in PCR?

Ex: DNA from epithelial cells of hands of individual performing assay can introduce foreign DNA into sample and all DNA present is amplified Meticulous attention to avoidance of DNA contamination is important Semi-quantitative assay at best Good at determining presence/absence of DNA sequence specific to a particular microbe directly from human specimen Not good at determining microbial load (infectious dose of patient, effectiveness of antimicrobic treatments) So real-time PCR developed

steps for PCR amplification of bacterial genes

First step to identify bacteria- use universal primer to amplify 16S rRNA region Next, sequence amplified DNA segments Third step, compare rDNA sequence of unidentified bacterial DNA fragment with known rDNA sequences in the genome database BLAST (Basic Local Alignment Search Tool) database sponsored by the National Center for Biotechnology Information, or NCBI and used to ID bacteria based on sequences BLAST will be used in this exercise DNA bacterial sequence with the best-fit match will provide ID of bacteria

Describe limitations as well as usefulness of end point PCR

Fluorescence and amplification not continually measured Traditional (endpoint) use agarose gels for detection of PCR amplification at final phase Limitations- time consuming, may take days, based on size discrimination, end point is variable from sample to sample

Define and describe the usefulness of melting curve analysis in real-time PCR

In the SYBR GREEN strategy, dye binds to amplified DNA and fluorescence increases Melting curve analysis can be used to check that the right fragments are being amplified Real-time machine monitors DNA synthesis and melting point of products at the end of amplification Melting point depends on composition All PCR products for a primer pair and sequence composition should have same melting temperatures unless there is contamination, mispriming, primer-dimer artifacts, other Check that all samples have a similar melting temperature to make sure no contamination At melting point, two strands will separate and fluorescence will decrease rapidly Rate of change of fluorescence vs time will peak at melting temperature Primer-dimer artifact will give a lower melting temperature peak (because short piece of DNA) and if peaks are not similar, there is contamination, mispriming, etc

Define and discuss how one can control for contamination in a PCR workflow lab

Melting curve can be analyzed to determine if there is the same melting point If different, may be contaminated Optimizing PCR reaction: annealing temperature of primers, concentration of Mg2+ in reaction, extension time, denaturing and annealing times, extension temperature, amount of template and polymerase Mg2+ is a cofactor for DNA polymerase Optimizing annealing temperature Primers have a calculated annealing temperature that must be confirmed practically Use a gradient cycler Temperature steps of 2 degrees Celsius above and below Optimizing Mg2+ concentration Fidelity of PCR depends on this Vary in steps of 0.5 mM Sometimes a compromise between yield and specificity necessary

PCR Amplification of Bacterial Genes

PCR can be used as a sensitive method for ID of bacteria that cannot be identified by traditional bacteriological methods 16S rRNA of bacteria has regions of conserved sequences and regions of sequence variation All bacteria share conserved regions of 16S rRNA Primer can detect 16S rRNA sequences that are conserved in all bacteria- universal primers Closely-related bacteria share portions of 16S rRNA sequence not found in other groups

Give at least one example of an application for high-fidelity/proofreading polymerase in PCR.

Phusion High-Fidelity DNA polymerase is a sd DNA-binding domain that is fused to a novel Pyrococcus-like enzyme that forms a unique high-performance polymerase called Phusion DNA polymerase that have the highest processivity Fidelity is a result of accurate replication of a desired template Ability to read template strand Select appropriate nucleoside triphosphate Insert correct nucleotide at the 3' primer terminus so W-C base pairing is maintained Some DNA polymerases possess a 3-5 exonuclease activity, proofreading, that takes out incorrectly incorporated mononucleotides and replace with correct one

technique used to selectively amplify a specific segment of DNA, mimics DNA replication, "molecular photocopier"

Polymerase Chain Reaction (PCR)

What is the advantage of NASBA-based techniques (TAS and branched DNA amplification) over the polymerase chain reaction (PCR)?

Possible to pick up mRNA in a dsDNA background without getting false positive results caused by genomic dsDNA

RNase H (digestion)

Primer 2 added and RNase H is an endonuclease that catalyzes cleavage of RNA via a hydrolytic mechanism (hybridization with target-specific oligonucleotide primer 2) Reverse transcriptase is used once again to create cDNA from these portions (reverse transcription)

Primer design for PCR

Primer design: should be 20 bases long G/C content should be 45-55% Annealing temperatures should be within 1 degree of each other 3'-most base should be G or C (strong hydrogen bonding of G/C) Primers must not base pair with each other or with themselves/form hairpins If a primer is self-complementary and can fold into a hairpin, the 3' end is base paired and cannot anneal to target DNA Primer can form dimer with itself or another primer which can be excellent but unwanted substrate for Taq polymerase Primers must avoid repetitive DNA regions Use annealing temperature that is 5 degrees Celsius lower than the melting temperature +Melting point can be calculated with Wallace formula: ™=4(C+G) + 2(A+T) degrees Celsius

Explain TaqMan strategy use in real-time PCR

Single-strand oligonucleotide probe designed to anneal to sequences within PCR and contains two fluorochromes 5' fluorochrome is called reporter and is usually 6-FAM (R) 3' fluorochrome is is TAMRA and is the quencher (Q) When probe intact and excited by light source, emission of reporter dye suppressed by quencher dye due to proximity When probe cleaved by 5' exonuclease activity of enzyme, distance increases and transfer of energy causes emission of reporter Probe designed to have higher ™ than primers During extension, probe must be 100% hybridized to the PCR product for assay to be accurate Both fluorochromes present on same molecule, no fluorescence will be observed Once Taq DNA polymerase initiates next round of synthesis, 5'-3' exonuclease activity of enzyme degrades TaqMan probe and releases the reporter fluorochrome from the quenched state Fluorescence recorded on continuous basis Probe can't be extended by polymerase

Describe RT-qPCR

Starting material is RNA and RNA is first transcripted into cDNA by reverse transcriptase from total or messenger RNA cDNA used as template for qPCR reaction

T7 DNA polymerase

T7 DNA polymerase extends these cDNA portions to amplify (generation of RNA transcript by action of T7 RNA polymerase)

Design of primers

T7 promoter at the 5' end with gene-specific sequence at the 3' end

When would one use Taq polymerase versus high-fidelity/proofreading polymerase in PCR?

Taq DNA polymerase lacks the 3-5 proof-reading activity commonly present in other polymerases Taq misincorporates 1 base in 10^4 Error distribution will be random proof-reading/high fidelity thermostably enzyme rather than Taq used when we need to clone amplified DNA because an individual molecule may harbor several mutations (use if errors matter) If you want to cut amplified product with restriction enzymes (has exonuclease) or screen for the presence/absence of pathogens or DNA products, use Taq (errors do not matter)

describe exponential process of PCR

The product of one cycle serves as the template for the next Amplification of template progresses at a rate of 2n N = number of cycles Products are called amplicons and the DNA segment being amplified is called the target Amplification rate is good only if the reaction is 100% efficient No reaction performed in a test tube is 100% efficient Modified equation for PCR amplification: Y=(1+E)n Where Y = degree of amplification N = number of cycles E = mean efficiency of amplification at each cycle; the percent of product from one cycle serving as template in the next

PCR: Kary Mullis able to invent from Thomas Brock's discovery of ___, which could withstand 100 degrees Celsius

Thermus aquaticus

qPCR

Threshold cycle value used for quantification- above which fluorescence can start to be seen Measured in exponential phase of reaction Can be used to calculate initial amount of template present due to reagents not being limited in the exponential phase (not the case for endpoint, plateau phase) If large template at start, few amplification cycles needed to accumulate enough product to give a fluorescence signal-- low CT If small amount present at start, more amplification needed for CT and it will be higher In both strategies, amount of fluorescence that indicates amplification recorded continuously Proportional to amount of PCR product in the reaction after each cycle (amplicons) Standard curve used for quantification If RNA template, reverse transcriptase converts to cDNA first Minimizes contamination because amplification and detection takes place in a single tube Needs special reagents/sophisticated insturmentation

What is the difference between transcription-based amplification and branched DNA technology?

Transcription-based amplification Isothermic, single-tube nucleic acid amplification system utilizing RNA polymerase and reverse transcriptase to rapidly amplify the target RNA, enabling simultaneous detection of multiple pathogenic organisms in a single tube Allows nucleic acid tests for blood screening with fewer steps and less time Branched DNA technology Signal amplification (not a target amplification) used to detect nucleic acid molecules, begins with a dish or some other solid support

qPCR applications

Viral quantitation Assessment of viral load Determination of treatment effectiveness (drug therapy) Quantitation of gene expression Array verification DNA damage measurement Quality control and assay validation Pathogen detection genotyping

products of PCR, piece of DNA or RNA that is the source and/or product of replication events

amplicons

critical parameters for PCR

concentration of DNA template and quality of DNA template, nucleotides, divalent cations (especially Mg2+), polymerase, error rate of polymerase (Taq, Vento exo, Pfu), primer design

software used to determine number of genomic DNA targets

copy number

not strictly ____ during early phase

doubling

Agarose gel results are obtained from the end point of the reaction.

endpoint PCR

Real-Time PCR: Principle of Quantification sensitivity throughput setup risk of contamination cost

exponential phase- depends on the initial sample quality very high: <5 copies of input target high-throughout possible easy, quick to perform. does not need post-pcr manipulation low very high

describe thermal cycler

instrument that rapidly makes the extreme temperature changes needed for PCR

what is PCR limited by?

integrity of starting DNA- 50 kb

what are slopes used for in PCR?

measure of efficiency, should be same with dilutions, problem if difference in slope and not parallel

what was PCR initially limited by?

primer synthesis and polymerase function issues

short strand of RNA or DNA (generally 18-22 bases) that serves as a starting point for DNA synthesis, required for DNA replication because DNA polymerases can only add new nucleotides to an existing strand of DNA

primers

what is bDNA technology used for?

quantification of HIV-1, HBV, and HCV

each strand of original DNA molecule, template for production of its counterpart (semiconservative replication)

template

template, digestion, and initial requirements for PCR

template does not need to be highly purified (boiled bacterial colony) PCR product can be digested with restriction enzymes, cloned, or sequence can amplify a single DNA molecule (like a single sperm)

no target products made until ____ cycle

third

cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, significantly above background fluorescence, at this level a detectable amount of amplicon product has been generated during early exponential phase of reaction In the first few cycles of real-time PCR, fluorescences = background levels and increases are not detectable despite accumulation of product Eventually, enough cumulates to give a detectable signa

threshold cycle

Describe components involved in nucleic acid sequence-based amplification (NASBA): Transcription-based amplification system (TAS)-

used to amplify RNA sequences, primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature

PCR can amplify a usable amount of DNA (_______) in about 2 hours

visible by gel eletrpohoresis