PCR and qPCR Fundamentals Quiz

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Questions and Answers

What is the main purpose of Polymerase Chain Reaction (PCR)?

To synthesize new DNA strands
To separate DNA molecules
To analyze DNA sequences
To amplify a DNA segment (correct)

What is the theoretical limit of DNA molecules that PCR can detect and amplify?

Ten DNA molecules
Ten million DNA molecules
One million DNA molecules
One DNA molecule (correct)

Why is contamination of samples a serious issue in PCR?

Due to the extreme sensitivity of PCR methods (correct)
Due to the requirement of specific buffer solutions
Due to the presence of DNA polymerase
Due to the need for high-quality DNA samples

What is the role of buffer in a PCR reaction?

To maintain the optimal pH for DNA polymerase activity (A) Signup and view all the answers

In the Livak method, how is the expression fold difference for Gene1 in flower tissue relative to leaf tissue calculated?

$2^{-(CT_{target} - CT_{reference}){sample} - (CT{target} - CT_{reference})_{calibrator}}$ (B) Signup and view all the answers

What is the formula to calculate the expression ratio (fold difference) in the Livak method?

Expression ratio (folds) = $2^{-(CT_{target} - CT_{reference}){sample} - (CT{target} - CT_{reference})_{calibrator}}$ (B) Signup and view all the answers

What is the purpose of normalizing the CT of the target to the reference in the Livak method?

To obtain the difference in CT values between the target and reference genes (A) Signup and view all the answers

What is the ΔΔCT in the Livak method?

The difference between the ΔCT of the test sample and the ΔCT of the calibrator (C) Signup and view all the answers

What is the purpose of the melting point analysis in qPCR?

To distinguish between amplified fragments (C) Signup and view all the answers

What is a disadvantage of End-Point PCR mentioned in the text?

Results are not expressed as numbers (D) Signup and view all the answers

What is the disadvantage of staining DNA in gel, as mentioned in the text?

It is not very quantitative (C) Signup and view all the answers

What is the disadvantage of qPCR mentioned in the text?

Requires relatively large amounts of DNA for detection (C) Signup and view all the answers

What is the main advantage of qPCR over traditional PCR with end-point detection?

Ability to monitor DNA synthesis after each cycle (A) Signup and view all the answers

Which type of PCR involves monitoring DNA synthesis after each cycle?

Real-time (qPCR) PCR (D) Signup and view all the answers

What is the role of Taq DNA polymerase in PCR?

Synthesizes new DNA strands (C) Signup and view all the answers

Which dye binds double stranded DNA and emits fluorescence in qPCR?

SYBR green (D) Signup and view all the answers

What is the main disadvantage of traditional PCR with end-point detection compared to qPCR?

Inability to monitor DNA synthesis after each cycle (C) Signup and view all the answers

Which method is commonly used for analyzing qPCR data?

Livak ΔΔCT method (C) Signup and view all the answers

What do reference genes in qPCR experiments help to normalize?

Expression of the target gene (C) Signup and view all the answers

What is the role of DNA probes in qPCR?

Detect DNA in the reaction (C) Signup and view all the answers

What is the main purpose of traditional PCR with end-point detection?

Amplify DNA for various applications (A) Signup and view all the answers

Which phase of DNA amplification in PCR involves exponential, linear, and plateau phases?

Amplification phase (A) Signup and view all the answers

What is the terminology used in qPCR to describe the value at which the fluorescence signal crosses the threshold?

Cycle threshold value (CT value) (D) Signup and view all the answers

What is the ideal application of qPCR?

Studying gene expression (B) Signup and view all the answers

Explain the process of calculating the expression fold difference for Gene1 in flower tissue relative to leaf tissue using the Livak method.

<ol> <li>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) $Expression ratio (folds) = 2^{-\Delta\Delta CT}$ $Fold Change = 2^{-\Delta\Delta CT} = 2 -((CT target - CT reference) in sample - (CT target - CT reference) in calibrator)$</li> </ol> Signup and view all the answers

What data is needed to be normalized to the reference gene in the Livak method?

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

How can the relative expression of Gene 1 in flower tissue be determined using qPCR data?

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

What is the formula to calculate the expression ratio (fold difference) in the Livak method?

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

What can be done to distinguish between amplified fragments in qPCR?

In qPCR, melting point analysis can be done to distinguish between amplified fragments. Signup and view all the answers

Explain the Livak method and how it is used to calculate the expression fold differences for Gene1 in flower tissue relative to leaf tissue.

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. Signup and view all the answers

What are the disadvantages of End-Point PCR mentioned in the text?

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. Signup and view all the answers

How is the relative expression of Gene 1 in flower tissue compared to leaf tissue determined using qPCR data?

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. Signup and view all the answers

What is the role of Actin in the qPCR experiments mentioned in the text?

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. Signup and view all the answers

What is the main purpose of the melting point analysis in qPCR?

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. Signup and view all the answers

Explain the process of DNA cloning and the role of restriction endonucleases in this process.

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. Signup and view all the answers

What are the three types of restriction endonucleases and how do they function?

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. Signup and view all the answers

What are the key steps involved in DNA/gene cloning?

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. Signup and view all the answers

Explain the process of selecting restriction enzymes for DNA cloning. What factors should be considered when choosing these enzymes?

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. Signup and view all the answers

What are the key features of cloning vectors and how are they used in gene cloning?

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. Signup and view all the answers

What is the pUC19 vector and why is it considered ideal for gene cloning and maintenance?

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. Signup and view all the answers

What are the three classes of vectors used in DNA cloning and what are their respective purposes?

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. Signup and view all the answers

Explain the process of ligation in DNA cloning and discuss the potential issues that may arise during this step.

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. Signup and view all the answers

How can DNA fragments be modified to generate sticky ends for sub-cloning genes using PCR?

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. Signup and view all the answers

What are the methods used to transfer recombinant DNA into a host organism for replication and maintenance?

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. Signup and view all the answers

What is the cutting frequency of restriction endonucleases and how can it be calculated?

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. Signup and view all the answers

What is the role of Type II restriction endonucleases in DNA work, and how do they cleave DNA?

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. Signup and view all the answers

What are the key characteristics of Type III restriction endonucleases and how do they function in DNA cleavage?

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. Signup and view all the answers

Discuss the significance of multiple cloning sites in cloning vectors and how they contribute to DNA cloning processes.

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. Signup and view all the answers

What is the significance of the origin of replication in cloning vectors, and how does it contribute to the maintenance of recombinant DNA?

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. Signup and view all the answers

Explain the difference between selectable markers and screenable markers in the context of identifying host cells harboring a vector in gene cloning.

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. Signup and view all the answers

What are some common antibiotics used for selecting cells containing the vector in gene cloning, and how do these antibiotics function?

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. Signup and view all the answers

How does the blue-white selection technology work in identifying bacteria that harbor the vector with an insert versus those without the insert?

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. Signup and view all the answers

What is the purpose of the LacZ gene as a screenable marker in gene cloning, and how does it function?

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. Signup and view all the answers

What is the process of blue-white selection using pUC vectors, and how does it help in identifying bacteria with the desired insert?

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. Signup and view all the answers

What is the procedure for ensuring that host cells carry the correct vector after transformation in gene cloning?

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. Signup and view all the answers

Explain the process of gel electrophoresis for determining the size of DNA fragments in gene cloning.

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. Signup and view all the answers

Are there alternative methods of identifying cells harboring the correct vector in gene cloning, and if so, what are they?

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

What is the significance of the blue-white selection technology in gene cloning, and how does it aid in the identification of bacteria with the desired insert?

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. Signup and view all the answers

How does the use of X-Gal and β-galactosidase in the blue-white selection technology help in identifying bacteria with the desired insert in gene cloning?

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. Signup and view all the answers

What is the role of agarose gel electrophoresis in gene cloning, and how does it contribute to the identification of DNA fragments?

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. Signup and view all the answers

What are selectable markers in gene cloning, and how are they used to identify host cells harboring the vector?

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. Signup and view all the answers

What are screenable markers in gene cloning, and how do they differ from selectable markers?

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. Signup and view all the answers

How is the LacZ gene used as a screenable marker in gene cloning?

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. Signup and view all the answers

What is the purpose of blue-white selection technology in gene cloning, and how does it work?

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. Signup and view all the answers

What chemical reaction occurs in the blue-white selection process using X-Gal, and what are the visible outcomes?

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. Signup and view all the answers

Aside from antibiotic resistance and screenable markers, what other method can be used to ensure host cells carry the correct vector after gene cloning?

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. Signup and view all the answers

How is the size of a DNA fragment determined using agarose gel electrophoresis?

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. Signup and view all the answers

What is the purpose of staining and visualizing DNA after agarose gel electrophoresis?

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. Signup and view all the answers

What is the role of the DNA marker in agarose gel electrophoresis, and how is it used?

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. Signup and view all the answers

What is the significance of subjecting the gel to electrophoresis at 50-100 volts for two hours?

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. Signup and view all the answers

Why is it important to load each DNA sample on a separate lane during agarose gel electrophoresis?

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. Signup and view all the answers

Besides agarose gel electrophoresis, what additional follow-up method can be used to ensure cells carry the correct vector after gene cloning?

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. Signup and view all the answers

What are the elements of protein structures?

Amino acids, peptide bonds, secondary structures, and tertiary structures (A) Signup and view all the answers

Why do proteins form the structures they have?

To achieve the most stable and energetically favorable conformation (C) Signup and view all the answers

What is the general structure of amino acids?

A central carbon bonded to an amino group, a carboxyl group, a hydrogen, and a side chain (R) (D) Signup and view all the answers

What type of bond forms between the 'C' from COO- of one amino acid and the N from another amino acid?

Peptide bond (A) Signup and view all the answers

What is the purpose of Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)?

To separate proteins based on their size (A) Signup and view all the answers

What is the primary principle behind Ion-exchange chromatography in protein separation?

Interaction of the protein with a charged matrix in the column (C) Signup and view all the answers

What is the key feature of the α/β barrel domain?

It is made of four β-αβ motifs interconnected by α helices (D) Signup and view all the answers

What is the main principle behind Gel/Size-exclusion chromatography in protein separation?

Movement of protein based on its size (D) Signup and view all the answers

What is the common feature of the helix-turn-helix motif?

Found in proteins that bind specific DNA sequences (B) Signup and view all the answers

What is the primary method used for protein separation in High-Pressure Liquid Chromatography (HPLC)?

Forced movement through a column containing a particular matrix (C) Signup and view all the answers

What is the primary principle behind Affinity Chromatography in protein separation?

Relies on the “affinity” of a given protein to another molecule (D) Signup and view all the answers

What is the main feature of the β-α-β motif?

Contains two parallel β strands connected by an α helix (D) Signup and view all the answers

What is the primary structure of a protein?

The amino acid sequence (B) Signup and view all the answers

What type of amino acids are typically found on the surface of proteins?

Amino acids with polar R groups (D) Signup and view all the answers

What is the secondary structure of a protein that is stabilized by intrachain hydrogen bonds and has an electric dipole?

α helix (B) Signup and view all the answers

Which class of proteins is typically water or lipid soluble and has functional roles?

Globular proteins (A) Signup and view all the answers

What are domains in proteins?

Independent, stable folding units containing multiple supersecondary structural elements (C) Signup and view all the answers

Which of the following is a characteristic of fibrous proteins?

They are usually elongated and insoluble in water (D) Signup and view all the answers

What is the role of β sheets in protein structure?

To stabilize the protein core (B) Signup and view all the answers

What is the directional structure of amino acid residues in polypeptide chains?

Free amino group at the N-terminus and a free carboxyl group at the C-terminus (B) Signup and view all the answers

What are the R groups of amino acids grouped based on?

Their properties (D) Signup and view all the answers

What is the tertiary structure of a protein?

Signup and view all the answers

What are the secondary protein structures?

α helices, β sheets, turns, and random coils (B) Signup and view all the answers

What stabilizes the α helix in proteins?

Intrachain hydrogen bonds (B) Signup and view all the answers

What is the function of Type I restriction endonucleases?

Cut DNA at random sites away from their recognition site (B) Signup and view all the answers

What is the primary purpose of the LacZ gene in gene cloning?

To enable blue-white selection to identify bacteria with the desired insert (B) Signup and view all the answers

What is the process of DNA/gene cloning involving a vector?

Separating a specific gene from a larger DNA and inserting it into a vector (C) Signup and view all the answers

What is the purpose of using a cDNA library in cloning a gene?

To clone a protein-encoding gene for which DNA sequence is unknown (D) Signup and view all the answers

What is the main advantage of using a cDNA library in cloning a gene compared to genomic DNA?

It contains only the expressed genes of an organism (C) Signup and view all the answers

What is the role of a probe in cloning a gene from a cDNA library?

To hybridize with the complementary DNA sequence of the gene of interest (B) Signup and view all the answers

What is the significance of isolating and characterizing the limonene synthase cDNA from spearmint?

It allows for the production of the catalytically active monoterpene cyclase (D) Signup and view all the answers

Which of the following primers can be used to amplify the cDNA encoding the polypeptide “FFM(X92)WMC”?

Forward: TT(T/C)TT(C/T)ATG & Reverse: TGGATGTG(C/T) (B) Signup and view all the answers

What is the significance of subjecting the gel to electrophoresis at 50-100 volts for two hours?

To ensure proper separation of DNA fragments based on size (A) Signup and view all the answers

What platform was used to sequence the gene inserted into the plasmid?

Sanger sequencing (D) Signup and view all the answers

What is the purpose of blue-white selection using pUC vectors in gene cloning?

To identify bacteria with the desired insert (D) Signup and view all the answers

What is the primary principle behind Ion-exchange chromatography in protein separation?

Separation based on charge differences (C) Signup and view all the answers

What is the main feature of the β-α-β motif?

It is a common secondary structure in proteins (B) Signup and view all the answers

What is the role of DNA probes in qPCR?

To bind to specific DNA sequences and allow their detection (B) Signup and view all the answers

What is the process of blue-white selection using pUC vectors, and how does it help in identifying bacteria with the desired insert?

It allows for identification of bacterial colonies with the desired insert based on color differences (D) Signup and view all the answers

What is the first step in building a cDNA library?

Obtaining total RNA/mRNA (C) Signup and view all the answers

What is the purpose of normalizing cDNA libraries?

To enrich for unstable or poorly expressed mRNA (D) Signup and view all the answers

What is the purpose of using methylated cDNA in lambda Zap cDNA synthesis kits?

To obtain a DNA probe for an 'unknown' protein-coding gene (D) Signup and view all the answers

What method was used to purify the 4S-limonene synthase protein?

Dye ligand chromatography, anion exchange chromatography, and hydrophobic interaction chromatography (C) Signup and view all the answers

What was done to the purified LimS protein to design degenerate primers for screening a cDNA library?

The protein was fragmented using Cyanogen Bromide (A) Signup and view all the answers

What was utilized to design three distinct oligonucleotide probes for screening a spearmint leaf cDNA library?

The internal amino acid sequences of the purified enzyme from spearmint oil glands (B) Signup and view all the answers

What was used to amplify DNA fragments (probes) from the cDNA library for screening?

PCR with forward and reverse primers based on the amino acid sequence of the LimS protein (A) Signup and view all the answers

What is the classical strategy for screening a cDNA library to isolate a full-length clone?

Screening the library with a probe (B) Signup and view all the answers

What is the process of obtaining a DNA probe for an 'unknown' protein-coding gene?

Obtaining purified protein from the target tissue, deducing DNA sequence for protein fragments, and designing PCR primers against the deduced cDNA (C) Signup and view all the answers

What is the purpose of ligating Poly T in building a cDNA library?

To prime the synthesis of cDNA (A) Signup and view all the answers

What is the role of screening the cDNA library with a probe specific for the gene of interest?

To isolate the full-length clone of the gene of interest (D) Signup and view all the answers

What is the first step in the classic approach of cloning a gene from a cDNA library?

Build a cDNA library from a target tissue (C) Signup and view all the answers

What is the process of screening a cDNA library?

Grow several million bacterial colonies from the cDNA library on plates (D) Signup and view all the answers

Which reference provides information about the cloning and functional characterization of β-phellandrene synthase from Lavandula angustifolia?

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

What is the focus of the lecture in Biol 366?

Cloning of βphellandrene synthase and S-linalool synthase from lavender (A) Signup and view all the answers

What is the primary purpose of lavender β-phellandrene synthase, LaβPHLS?

Producing β-phellandrene as the major product using GPP and NPP as substrates (C) Signup and view all the answers

Which method is used for cloning genes involving de novo assembly of contigs representing mRNA?

Transcriptome sequencing (C) Signup and view all the answers

What is the purpose of the GST-GSH system in protein purification?

To remove/purify fusion proteins from a protein mixture (D) Signup and view all the answers

What are the important elements in an expression vector used for producing recombinant proteins in E. coli?

Promoter, operator, and selectable marker (C) Signup and view all the answers

What is the role of the RecA gene when cloned into an expression vector?

Producing large amounts of the RecA protein in induced bacterial cells (B) Signup and view all the answers

What is the purpose of affinity chromatography in protein purification?

To purify recombinant proteins (C) Signup and view all the answers

What is the function of the GST tag in protein purification?

To facilitate purification of the tagged fusion protein (A) Signup and view all the answers

What is the purpose of using RNA-Seq for cloning lavender S-linalool synthase?

To involve de novo assembly of contigs representing mRNA (D) Signup and view all the answers

What is the major product produced by Lavandula angustifolia EST database gene LaβPHLS?

β-phellandrene (C) Signup and view all the answers

What is the purpose of using an expression vector with a promoter, operator, and selectable marker for producing recombinant proteins in E. coli?

To regulate gene expression and enable selection of transformed cells (A) Signup and view all the answers

What is the primary role of RNA-Seq in cloning genes?

De novo assembly of contigs representing mRNA (D) Signup and view all the answers

Flashcards

PCR (Polymerase Chain Reaction)

A technique that amplifies specific DNA sequences using repeated cycles of denaturation, annealing, and extension.

qPCR (Quantitative PCR)

A type of PCR that allows real-time monitoring of DNA synthesis and quantification of DNA targets.