PCR and qPCR Fundamentals Quiz

To amplify a DNA segment

One DNA molecule

Due to the extreme sensitivity of PCR methods

To maintain the optimal pH for DNA polymerase activity

$2^{-(CT_{target} - CT_{reference}){sample} - (CT{target} - CT_{reference})_{calibrator}}$

Expression ratio (folds) = $2^{-(CT_{target} - CT_{reference}){sample} - (CT{target} - CT_{reference})_{calibrator}}$

To obtain the difference in CT values between the target and reference genes

The difference between the ΔCT of the test sample and the ΔCT of the calibrator

To distinguish between amplified fragments

Results are not expressed as numbers

It is not very quantitative

Requires relatively large amounts of DNA for detection

Ability to monitor DNA synthesis after each cycle

Real-time (qPCR) PCR

Synthesizes new DNA strands

SYBR green

Inability to monitor DNA synthesis after each cycle

Livak ΔΔCT method

Expression of the target gene

Detect DNA in the reaction

Amplify DNA for various applications

Amplification phase

Cycle threshold value (CT value)

Studying gene expression

1. Calculate the average $CT$ value for each gene in both flower and leaf tissue. 2) Normalize the $CT$ of target to reference (obtain $\Delta CT$) in sample (flower) and calibrator (leaf) $\Delta CT (sample) = CT (target in sample) - CT (reference in sample)$ $\Delta CT (calibrator) = CT (target in calibrator) - CT (reference calibrator)$ 3) Calculate the difference between the $\Delta CT$ of the test sample and the $\Delta CT$ of the calibrator, also called $\Delta\Delta CT$ $\Delta\Delta CT = \Delta CT (sample) - \Delta CT (calibrator)$ 4) Calculate the expression ratio (fold difference) $Expression ratio (folds) = 2^{-\Delta\Delta CT}$ $Fold Change = 2^{-\Delta\Delta CT} = 2 -((CT target - CT reference) in sample - (CT target - CT reference) in calibrator)$

The $CT$ values of the target gene and the reference gene need to be normalized in the Livak method.

The relative expression of Gene 1 in flower tissue can be determined by calculating the expression fold difference using the Livak method.

The formula to calculate the expression ratio (fold difference) in the Livak method is $Expression ratio (folds) = 2^{-\Delta\Delta CT}$.

In qPCR, melting point analysis can be done to distinguish between amplified fragments.

The Livak method involves the following steps: 1) Calculate the average CT value for each gene in both flower and leaf tissue. 2) Normalize the CT of the target to the reference (obtain ΔCT) in the sample (flower) and calibrator (leaf). 3) Calculate the difference between the ΔCT of the test sample and the ΔCT of the calibrator, also called ΔΔCT. 4) Calculate the expression ratio (fold difference) using the formula $2^{-\Delta\Delta CT} = 2 - ((CT_{target} - CT_{reference}){sample} - (CT{target} - CT_{reference})_{calibrator})$. This method is used to determine the relative expression of a target gene in different tissues or under different conditions.

The disadvantages of End-Point PCR mentioned in the text include: non-automated process, post-PCR processing needed, results not expressed as numbers, poor precision, low sensitivity, and size-based discrimination only.

The relative expression of Gene 1 in flower tissue compared to leaf tissue is determined using the Livak method, which involves calculating the expression fold differences using the formula $2^{-\Delta\Delta CT} = 2 - ((CT_{target} - CT_{reference}){sample} - (CT{target} - CT_{reference})_{calibrator})$. This method normalizes the CT values of the target gene to the reference gene and calculates the fold difference in expression.

Actin is used as a reference gene in the qPCR experiments mentioned in the text. It is used to normalize the CT values of the target gene (Gene 1) and to calculate the relative expression of Gene 1 in flower tissue compared to leaf tissue.

The main purpose of the melting point analysis in qPCR is to distinguish between amplified fragments. It helps in identifying the specific products of the PCR reaction and differentiating them from non-specific amplification products.

DNA cloning involves separating a specific gene or DNA fragment from a larger DNA, inserting it into a vector (plasmid or carrier DNA), introducing the construct into a host cell, and replicating the DNA in the cell. Restriction endonucleases (RE) recognize specific nucleotide sequences and cut the DNA at those sites, facilitating the process of separating the desired DNA fragment from the larger DNA molecule.

The three types of restriction endonucleases are Type I, Type II, and Type III. Type I enzymes cut DNA at a random site that is different and away (hundreds of bases) from their recognition site. Type II enzymes recognize specific nucleotide sequences and cut the DNA at those sites. Type III enzymes recognize specific nucleotide sequences and cut the DNA a short distance (about 25 base pairs) from their recognition site.

The key steps involved in DNA/gene cloning include separating a specific gene or DNA fragment from a larger DNA, inserting it into a vector (plasmid or carrier DNA), introducing the construct into a host cell, and replicating the DNA in the cell.

When selecting restriction enzymes for DNA cloning, factors such as the size and type of the DNA fragments, the presence of specific recognition sites, and the compatibility of the enzyme buffer systems should be considered. Other factors include the ability to generate compatible cohesive (sticky) or blunt ends for ligation, as well as the cutting frequency of the enzyme, which can be calculated using the equation Y = 4n, where Y is the cutting frequency and n is the number of recognition sites in the DNA sequence.

Cloning vectors are DNA "vehicles" capable of self-replication and contain features such as origin of replication, selectable marker, screenable marker, and multiple cloning site. These vectors are used in gene cloning by providing the necessary elements for the insertion, selection, and replication of the target gene within a host organism.

The pUC19 vector is a commonly used cloning vector that contains features such as a high copy number origin of replication, multiple cloning sites, and selectable markers for ampicillin resistance and lacZα complementation. It is considered ideal for gene cloning and maintenance due to its versatility, high efficiency of transformation, and ability to easily screen for recombinant clones.

The three classes of vectors used in DNA cloning are housekeeping vectors, expression vectors, and delivery vectors. Housekeeping vectors are used for general cloning and maintenance, expression vectors are designed for the expression of target genes, and delivery vectors are used to transfer DNA into a host organism.

Ligation, catalyzed by DNA ligases, joins DNA ends at strand breaks or nicks, but self-ligation of the vector or formation of concatemers may occur. During ligation, it is important to optimize the ratio of insert to vector to minimize self-ligation and ensure efficient ligation of the target gene. Additionally, the use of high-quality DNA ligase and proper reaction conditions is crucial to prevent potential issues during ligation.

DNA fragments can be modified to generate sticky ends by designing PCR primers with overhang sequences that complement the desired sticky ends. During PCR amplification, the overhang sequences are incorporated into the DNA fragments, creating complementary sticky ends that can facilitate the ligation of the target gene into a vector.

Recombinant DNA can be transferred into a host organism for replication and maintenance using methods such as heat shock, electroporation, or conjugation. These methods allow for the efficient uptake of foreign DNA by the host organism, leading to the replication and expression of the recombinant genes.

The cutting frequency of restriction endonucleases depends on sequence composition and can be calculated using the equation Y = 4n, where Y represents the cutting frequency and n represents the number of recognition sites in the DNA sequence. This equation allows for the estimation of the frequency at which a specific restriction enzyme will cut the DNA at its recognition sites.

Type II restriction endonucleases are commonly used in DNA work to cleave DNA within their recognition sites. These enzymes recognize palindromic sequences that are 4–8 nucleotides long and cleave the DNA at specific positions within the recognition site, generating cohesive (sticky) or blunt ends that can be used for DNA cloning and manipulation.

Type III restriction endonucleases recognize two inversely oriented non-palindromic sequences and cut DNA about 20-30 base pairs after the recognition site. Unlike Type II restriction endonucleases, Type III enzymes cleave DNA at a distance from their recognition sites, allowing for the production of DNA fragments with defined overhangs that can facilitate DNA cloning and recombinant DNA technology.

Multiple cloning sites in cloning vectors provide a range of unique restriction enzyme recognition sequences, allowing for the efficient insertion of target DNA fragments into the vector. These sites enhance the versatility and flexibility of DNA cloning processes by enabling the use of various restriction enzymes to generate compatible ends for ligation, ultimately facilitating the construction of recombinant DNA molecules.

The origin of replication in cloning vectors is essential for the autonomous replication of recombinant DNA within a host organism. This feature ensures the propagation and maintenance of the inserted DNA sequences during cell division, allowing for the stable inheritance and amplification of the recombinant genes. Additionally, the origin of replication plays a crucial role in the production of multiple copies of the recombinant DNA, which is important for various applications in molecular biology.

Selectable markers are genes that allow for the selection and survival of transformed cells under specific conditions, typically by conferring resistance to certain antibiotics. Screenable markers, on the other hand, are genes encoding proteins that cause a visible change in cell appearance, such as producing a color or fluorescence, allowing for easy identification of cells carrying the recombinant plasmid.

Common antibiotics used are ampicillin, kanamycin, and tetracycline. Cells containing the vector become resistant to the corresponding antibiotic and can survive, while cells lacking the resistance gene do not grow in the presence of the antibiotic.

Bacteria harboring the vector with the insert DNA remain white in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), while bacteria harboring the empty vector (without the insert DNA) turn blue in the presence of X-Gal. This allows for the selection of bacteria that harbor the vector with the desired insert.

The LacZ gene encodes β-galactosidase in bacteria harboring pUC vectors. When the LacZ gene is intact, it allows the production of β-galactosidase, leading to a color change in the cells. If the LacZ gene is disrupted by a DNA fragment, the bacteria stay white. This allows for the easy identification of cells carrying the recombinant plasmid.

Bacterial colonies grown on agar plates containing X-Gal and an inducer of β-galactosidase turn blue when they harbor a functional lacZ gene. This allows for the identification of bacteria with the desired insert, as they will remain white in the presence of X-Gal.

After growing resistant cells on antibiotic, the plasmid is extracted from the cells, digested with restriction enzymes, and the digested DNA is run on a gel to look for the cloned gene by size. Follow-up sequencing may be performed if needed to confirm the presence of the desired DNA insert.

The DNA samples are loaded onto separate lanes of an agarose gel, along with a DNA marker of known sizes on one lane. The gel is then subjected to electrophoresis at a specific voltage for a certain duration. After staining and visualization, DNA fragments of the same size migrate at the same velocity, allowing for the determination of their sizes.

Yes, alternative methods include fluorescent tagging of the vector, PCR screening, and sequencing of the inserted DNA to confirm its presence.

The blue-white selection technology allows for the selection of bacteria that harbor the vector with the desired insert by differentiating them from those with the empty vector. This aids in the efficient identification of transformants with the desired DNA insert.

The presence of a functional lacZ gene in bacteria harboring the vector with the desired insert results in white colonies, while the absence or disruption of the lacZ gene leads to blue colonies. This allows for the visual identification of bacteria with the desired insert.

Agarose gel electrophoresis is used to separate DNA fragments based on size, allowing for the visualization and determination of the sizes of the DNA fragments. This aids in the identification of the cloned gene by size and the confirmation of the presence of the desired DNA insert.

Selectable markers are genes introduced into a cell that either permit the growth of the cell or kill it under certain conditions. Commonly used selectable markers include ampicillin, kanamycin, and tetracycline. Cells containing the vector become resistant to the corresponding antibiotic, allowing them to survive, while cells lacking the resistance gene do not grow in the presence of the antibiotic.

Screenable markers are genes encoding proteins that cause a visible change in cell appearance, such as producing a color or making the cell fluoresce. Unlike selectable markers, cells with or without the screenable marker gene are not harmed. The cells carrying the recombinant plasmid can be easily identified by the colored or fluorescent colonies they produce.

The LacZ gene, when intact, allows the production of β-galactosidase in bacteria harboring pUC vectors. If the LacZ gene is disrupted by a DNA fragment, it will not produce a functional protein, causing the bacteria harboring pUC with a disrupted LacZ gene to appear white.

The purpose of blue-white selection technology is to select for bacteria that harbor the vector with the insert DNA. Bacteria harboring the empty pUC vector turn blue in the presence of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal), while bacteria harboring the pUC vector containing the insert DNA remain white.

β-Galactosidase cleaves X-Gal to produce galactose and 5-bromo-4-chloro-3-hydroxyindole, which dimerizes and is oxidized to an insoluble blue compound. Bacterial colonies grown on agar plates containing X-Gal and an inducer of β-galactosidase turn blue if they harbor a functional lacZ gene.

After growing resistant cells on antibiotic and extracting the plasmid from cells, researchers can digest the plasmid with restriction enzymes, run the digested DNA on a gel, and look for the cloned gene by size. This can be followed up with sequencing if needed.

DNA fragments of the same size migrate at the same velocity when placed in an electric field during agarose gel electrophoresis. By comparing the migration of sample DNA with a DNA marker of known sizes, the size of the DNA fragments can be determined.

Staining and visualizing DNA allows researchers to observe the separation of DNA fragments based on size and confirm the presence of the desired DNA fragments.

The DNA marker is a mix of DNA segments of known sizes, and it is loaded on one lane of the gel. It serves as a reference to determine the size of the sample DNA fragments by comparison.

Subjecting the gel to electrophoresis at 50-100 volts for two hours allows the DNA fragments to migrate through the gel, separating based on size.

Loading each DNA sample on a separate lane ensures that the DNA fragments from different samples do not mix and can be individually analyzed based on their sizes.

In addition to agarose gel electrophoresis, researchers can follow up with sequencing if needed to ensure that the cells carry the correct vector after gene cloning.

Amino acids, peptide bonds, secondary structures, and tertiary structures

To achieve the most stable and energetically favorable conformation

A central carbon bonded to an amino group, a carboxyl group, a hydrogen, and a side chain (R)

Peptide bond

To separate proteins based on their size

Interaction of the protein with a charged matrix in the column

It is made of four β-αβ motifs interconnected by α helices

Movement of protein based on its size

Found in proteins that bind specific DNA sequences

Forced movement through a column containing a particular matrix

Relies on the “affinity” of a given protein to another molecule

Contains two parallel β strands connected by an α helix

The amino acid sequence

Amino acids with polar R groups

α helix

Globular proteins

Independent, stable folding units containing multiple supersecondary structural elements

They are usually elongated and insoluble in water

To stabilize the protein core

Free amino group at the N-terminus and a free carboxyl group at the C-terminus

Their properties

α helices, β sheets, turns, and random coils

Intrachain hydrogen bonds

Cut DNA at random sites away from their recognition site

To enable blue-white selection to identify bacteria with the desired insert

Separating a specific gene from a larger DNA and inserting it into a vector

To clone a protein-encoding gene for which DNA sequence is unknown

It contains only the expressed genes of an organism

To hybridize with the complementary DNA sequence of the gene of interest

It allows for the production of the catalytically active monoterpene cyclase

Forward: TT(T/C)TT(C/T)ATG & Reverse: TGGATGTG(C/T)

To ensure proper separation of DNA fragments based on size

Sanger sequencing

To identify bacteria with the desired insert

Separation based on charge differences

It is a common secondary structure in proteins

To bind to specific DNA sequences and allow their detection

It allows for identification of bacterial colonies with the desired insert based on color differences

Obtaining total RNA/mRNA

To enrich for unstable or poorly expressed mRNA

To obtain a DNA probe for an 'unknown' protein-coding gene

Dye ligand chromatography, anion exchange chromatography, and hydrophobic interaction chromatography

The protein was fragmented using Cyanogen Bromide

The internal amino acid sequences of the purified enzyme from spearmint oil glands

PCR with forward and reverse primers based on the amino acid sequence of the LimS protein

Screening the library with a probe

Obtaining purified protein from the target tissue, deducing DNA sequence for protein fragments, and designing PCR primers against the deduced cDNA

To prime the synthesis of cDNA

To isolate the full-length clone of the gene of interest

Build a cDNA library from a target tissue

Grow several million bacterial colonies from the cDNA library on plates

Demissie ZA, Sarker L, and Mahmoud SS (2011). Cloning and functional characterization of β-phellandrene synthase from Lavandula angustifolia. PLANTA 233: 685-696

Cloning of βphellandrene synthase and S-linalool synthase from lavender

Producing β-phellandrene as the major product using GPP and NPP as substrates

Transcriptome sequencing

To remove/purify fusion proteins from a protein mixture

Promoter, operator, and selectable marker

Producing large amounts of the RecA protein in induced bacterial cells

To purify recombinant proteins

To facilitate purification of the tagged fusion protein

To involve de novo assembly of contigs representing mRNA

β-phellandrene

To regulate gene expression and enable selection of transformed cells

De novo assembly of contigs representing mRNA