PCR [Complete]

The number of amplicons = [EXAM 2 QUESTION]

*2^N+1* where *N = the number of cycles* The +1 is there to account for the original sample

What is the optimal temperature for *thermostable DNA polymerases* for *extension*?

*72°C*

How does uracil‐N‐glycosylase work?

*Cleaves each post-PCR contaminant sequence at the Uracil*, rendering the small fragments *useless* and unable to cause problems

What would one do for PCR if *starting* the procedure with *RNA*?

*Convert RNA → DNA* by the *use* of *Reverse Transcriptase*

Every cycle of PCR results in:

*Double the amount* of the original

PCR Amplification:

*Initial* denaturation → *Denature DNA* → *Anneal* primer → *Extend* primer [IDAE]

Most *commonly* used *Thermostable Polymerase*:

*Taq*; Thermus aquaticus

What is the main *goal* of *Primer Design*:

*Target Specificity*

This piece of equipment aids in automation of PCR by *changing the temperatures in a block or chamber holding the samples*:

*Thermal Cycler*

Why is qPCR using SyBr Green a popular method?

*Very cheap*

Peforming PCR [Overview]:

1. Assemble a reaction mix containing all components necessary for DNA synthesis. 2. Subject the reaction mix to an amplification program. 3. Analyze the product of the PCR reaction (the amplicon).

What is the term for the final PCR Product?

Amplicon

If *reagent blank* comes up as *positive*, what occured?

Contamination

Viral copy number quantification *Example*:

Ct[control] = 23.7 Ct[sample] = 21.6 Control Concentration = 125,000/mL 1.) Does W4047 have more or less HBV than the control? The sample has more HBV than the control because the threshold was crossed in fewer cycles. [I.e. Input amount is inversely proportion to threshold. The threshold cycle was smaller than the control thus there was more virus present] 2.) What is the Viral Load? Ct[23.7] - Ct[21.6] = 2.1 2^2.1 = 4.287 [Use base 2 because PCR is exponentially logarithmic] Thus, there is 4.287 fold more virus in specimen 4.287 x 125,000/mL = 535,875 viral load per mL

What is the *Threshold Cycle*?

Cycle at which fluorescence crosses the threshold fluorescence level

DNA Polymerase from E. coli would be heated and killed every cycle of the PCR reaction. This problem led to the production of:

DNA Polymerase from Thermus aquaticus (Taq). It is heat resistant.

What is unique about the PCR reaction tubes?

Extremely small and have special *thin-walled* tubes that can allow the internal temperatures to change rapidly.

What allows for the PCR product to be *quantifiable*?

Grows in an *exponential* fashion, *Doubling at each cycle*

An *increase* in *GC content* will result in the need of a:

Higher Tm [Melting Temp] [GC has 3 hydrogen bonds (AT has 2) and thus requires a higher temp to melt]

This may occur due to *NON-specific hybridization* of *primers*?

Misprimes

What problem with PCR requires the use of a *sequestering enzyme* to avoid?

Misprimes

Thermal Melt Curves can also be used to detect:

Mutations in cancers

Primer‐directed in vitro enzymatic reaction for the massive amplification (production) of a specific DNA fragment is the *definition of*:

Polymerase Chain Reaction

This may occur due to *hybridization of primers* to *EACH OTHER*:

Primer Dimers

What determines the *specificity of the PCR reaction*? [Exam question]

Primers

*Pfu*, another type of thermostable polymerase, is useful in conjunction with Taq because of it's _____________ abilities.

Proofreading

No product should be present in the?

Reagent Blank

This *MUST* always be included with each PCR run to *assess contamination*?

Reagent Blank

Purpose of PCR product cleanup?

Removes all reaction components from PCR product as well as misprimes and primer dimers. Required before many subsequent steps like DNA sequencing.

What makes cDNA? [Test Question]

Reverse Transcriptase

In terms of qPCR, describe the chemistries used:

SyBr green; Dye-based TaqMan; Hybridization based

Tm =

The temperature at which a piece of DNA will associate or disassociate from its complementary sequence during Annealing.

Input amount is *inversely proportional* to?

Threshold Cycle. [The more DNA you input in the beginning, the less time it takes to reach threshold cycle[

The *most dangerous* contaminant of PCR is:

a *PCR product from a previous PCR reaction*

Examples of qPCR detection systems:

∙ *Cepheid Smart Cycler* ∙ *Roche LightCycler* ∙ *Applied Biosystems 9600 Well Plate*

What MUST be used as a *template* for *PCR*?

∙ *DNA* [ds or ss] ∙ Should be as high quality/molecular weight as possible and as pure as possible

qPCR Detection Chemistries:

∙ *DNA‐specific dyes*: - *SyBr green* ∙ *Hybridization Probes*: - *Cleavage-based*; *TaqMan*

Denaturation:

∙ *Denatures DNA* ∙ *95°C*

Contamination Control:

∙ *Physical Separation*: - Air locks, positive air flow/ PCR hoods with UV ∙ uracil‐N‐glycosylase ∙ 10% bleach

uracil‐N‐glycosylase overview:

∙ *Problem: Essentially all PCR contamination is derived from previous PCR reactions* ∙ Solution: Add dUTP + uracil‐Nglycosylase. ∙ Place at 50°C for 2 minutes and it will cut at every Uracil base.

PCR Product Cleanup Methods:

∙ *Solid‐phase isolation of PCR product* [Ex: *Spin Columns*] ∙ *Small Sephadex G‐50 columns for the microfuge*

Quantitative PCR [qPCR]

∙ 1) Relative fluorescence in each PCR tube at the end of each cycle is measured and plotted ∙ 2) A threshold level of fluorescence is determined based on signal and background. ∙ 3) *Input* (starting DNA amount) *is inversely proportional to this "threshold" cycle*. [cycle at which fluorescence crosses the threshold fluorescence level]

Viral copy number quantification:

∙ 2^[Ct[control] - Ct[sample]]

Annealing:

∙ Anneals primer to template ∙ 5°C below the Tm [Melting Temp] for 20-30 seconds. ∙ Usually 50-68°C

Deoxynucleotide Triphosphates (dNTPs):

∙ Building blocks for the reaction ∙ Same as what is used in DNA replication [dATP, dGTP, dCTP, dTTP] ∙ Usually come in the kit *all included in a Master Mix*

PCR Step 2:

∙ Denature ∙ Anneal ∙ Extension [DAE]

Extension:

∙ Extends primer [Speeds up DNA Polymerase] ∙ *72°C* ∙ Polymerizes 1 kb/min at this temp

Left-To-Right primer?

∙ Forward Primer ∙ Moves 5' to 3'

PCR Step 1:

∙ Initial Denaturation

Real-Time Quantitative PCR (qPCR):

∙ PCR product *grows in an exponential fashion* [*doubling at each cycle=what makes is quantifiable*] ∙ PCR signal is observed as an exponential curve with a lag phase, a log phase, a linear phase, and a stationary phase.

*Principle* of qPCR using *SyBr Green*:

∙ PCR reaction contains Sybr Green ∙ *Very Cheap Method of qPCR* ∙ Binds minor groove of doublestranded DNA ∙ Do not need a special primer/probe set for every gene

An example of physical separation to prevent contamination when doing PCR is:

∙ Pre-PCR & Post-PCR rooms

PCR Primers: [Oligonucleotides]

∙ Primers are single‐stranded 18-30 base DNA fragments *complementary* to sequences flanking the region to be amplified. ∙ Primers determine the specificity of the PCR reaction. ∙ The distance between the primer binding sites will determine the size of the PCR product

Reverse‐Transcriptase PCR

∙ RNA cannot be amplified directly, but must first be converted to complementary DNA ∙ *RNA is converted to complementary DNA [cDNA] with the enzyme Reverse Transcriptase* ∙ The cDNA is used as a template for PCR ∙ Screen and monitor viral loads for HIV, HCV

Right-To-Left primer?

∙ Reverse Primer ∙ Moves 5' to 3'

Initial Denaturation:

∙ Splits template DNA strands ∙ 95°C for 3-15 min

Real‐Time or Quantitative PCR (qPCR) overview:

∙ Standard PCR with an *added probe or dye* to *generate a fluorescent signal from the product*. ∙ *Detection of signal in real time allows quantification of starting material*. ∙ Performed in specialized thermal cyclers with fluorescent detection systems.

Reverse Transcription process:

∙ Start with RNA [mRNA] ∙ *Can anneal poly dT primer against the polyA tail of RNA* OR ∙ *Anneal Random Hexamer* [Add Each base and reverse transcriptase begins random sequencing] ∙ Put at 42°C to allow Rev. Transcriptase to become active ∙ complementerary DNA strand is manaufactured.

qPCR; TaqMan: [Based on 3' to 5' exonuclease something something] [Test Question]

∙ TaqMan Probe is joined together with a *reporter dye* and a *quencher* ∙ When probe is intact, the quencher will block the reporter dye from fluorescing ∙ When the bond between the reporter dye and quencher is broken, the reporter dye is far enough away from the quencher that it will not block it from fluorescing ∙ This results in fluorescence being emitted and detected in the real-time /quantitative PCR process.

Interpretation of the PCR Results:

∙ The PCR product should be of the expected size. ∙ *No product* should be *present in* the *reagent blank*. ∙ *A reagent blank (negative/no DNA control MUST be included with each PCR run to assess contamination)* ∙ *Misprimes* may occur due to nonspecific hybridization of primers. ∙ *Primer dimers* may occur due to hybridization of primers to each other.

qPCR Detection Systems:

∙ Thermal cyclers with fluorescent detection and specialized software ∙ PCR reaction takes place in optically clear plates, tubes, or capillaries

How to *Avoid Misprimes*?

∙ Use a *Sequestered Enzyme* that requires an initial heat activation [Hot Start] ∙ Has an inactivating antibody bound to the enzyme

Thermal Melt Curve:

∙ With SyBr Green a Melt Curve analysis can be performed ∙ Each DNA fragment has a unique melting profile ∙ As the temperature of the PCR mix is slowly heated the dsDNA becomes ssDNA and thus SyBr green is released and no longer fluoresces ∙ Take the derivative of the change in fluorescence/derivative of the temperature = melt curve

What constitutes the "master mix"?

∙ dATP ∙ dGTP ∙ dCTP ∙ dTTP

A Standard Reverse Transcriptase Reaction:

∙ dATP, dCTP, dGTP, dTTP (dNTPs) ∙ Reverse Transcriptase & RT Reaction Buffer ∙ *poly dT or random Hexamer Primer* ∙ RNase Inhibitor ∙ *RNA for use as a template* (up to 5 ug)

Standard PCR Reaction Mix:

∙ dNTPs ∙ KCL, Tris pH 8.4, MgCl₂ ∙ DNA Taq polymerase ∙ Primer ∙ DNA