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Questions and Answers
What is a significant advantage of using SAGE technology?
What is a significant advantage of using SAGE technology?
RNA-seq requires prior knowledge of the genome to be effective.
RNA-seq requires prior knowledge of the genome to be effective.
False
Which method is used to study alternative splicing events associated with diseases?
Which method is used to study alternative splicing events associated with diseases?
RNA-seq
SAGE requires sequencing more than _____ tags per sample.
SAGE requires sequencing more than _____ tags per sample.
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Match the following technologies with their advantages:
Match the following technologies with their advantages:
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Which of the following is NOT a disadvantage of SAGE?
Which of the following is NOT a disadvantage of SAGE?
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Single-cell RNA-seq is a technique mainly used for studying individual cells to understand biological diversity.
Single-cell RNA-seq is a technique mainly used for studying individual cells to understand biological diversity.
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In RNA-seq, what component of RNA is traditionally sequenced?
In RNA-seq, what component of RNA is traditionally sequenced?
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Which of the following is an advantage of Reverse Transcriptase PCR?
Which of the following is an advantage of Reverse Transcriptase PCR?
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The main purpose of Differential Display is to compare two RNA samples to identify novel genes.
The main purpose of Differential Display is to compare two RNA samples to identify novel genes.
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What does RT-PCR stand for?
What does RT-PCR stand for?
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In Reverse Transcriptase PCR, _____ is used to produce cDNA from mRNA.
In Reverse Transcriptase PCR, _____ is used to produce cDNA from mRNA.
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Which of the following describes a disadvantage of open-ended technology?
Which of the following describes a disadvantage of open-ended technology?
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Which of the following is NOT an application of DDRT-PCR?
Which of the following is NOT an application of DDRT-PCR?
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DDRT-PCR can be utilized for analyzing gene expression during specific phases of the cell cycle.
DDRT-PCR can be utilized for analyzing gene expression during specific phases of the cell cycle.
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Match the PCR technique with its correct description:
Match the PCR technique with its correct description:
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What is the purpose of colony PCR in the screening process?
What is the purpose of colony PCR in the screening process?
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Real Time PCR can produce results by measuring fluorescence after amplification is complete.
Real Time PCR can produce results by measuring fluorescence after amplification is complete.
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Name one type of differentiation that DDRT-PCR can help identify genes for.
Name one type of differentiation that DDRT-PCR can help identify genes for.
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DDRT-PCR can identify genes related to __________, which are essential for cell proliferation.
DDRT-PCR can identify genes related to __________, which are essential for cell proliferation.
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What is one of the potential artifacts introduced during open-ended technology processes?
What is one of the potential artifacts introduced during open-ended technology processes?
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End repair or A-tailing is performed after ligation to ensure effective integration of cDNA into vectors.
End repair or A-tailing is performed after ligation to ensure effective integration of cDNA into vectors.
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Match the following applications of DDRT-PCR with their descriptions:
Match the following applications of DDRT-PCR with their descriptions:
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What is precloning?
What is precloning?
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What is one advantage of using DDRT-PCR in cell differentiation studies?
What is one advantage of using DDRT-PCR in cell differentiation studies?
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The process of using labeled probes specific to the target cDNA is known as _____ .
The process of using labeled probes specific to the target cDNA is known as _____ .
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Match the following methodologies with their descriptions:
Match the following methodologies with their descriptions:
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DDRT-PCR cannot reveal changes in gene expression during cell cycle arrest.
DDRT-PCR cannot reveal changes in gene expression during cell cycle arrest.
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Which of the following factors should be considered when selecting a vector?
Which of the following factors should be considered when selecting a vector?
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The ratio of cDNA insert to vector in a ligation protocol is typically 5:1.
The ratio of cDNA insert to vector in a ligation protocol is typically 5:1.
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What is the role of antibiotic resistance in vector selection?
What is the role of antibiotic resistance in vector selection?
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What can DDRT-PCR help identify in the context of cancer research?
What can DDRT-PCR help identify in the context of cancer research?
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DDRT-PCR can be utilized to identify drug targets and mechanisms of drug resistance.
DDRT-PCR can be utilized to identify drug targets and mechanisms of drug resistance.
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What is analyzed to gain insight into why some cells are sensitive to drug action?
What is analyzed to gain insight into why some cells are sensitive to drug action?
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DDRT-PCR has been applied in laboratories to identify genes involved in __________.
DDRT-PCR has been applied in laboratories to identify genes involved in __________.
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Which of the following actions utilize DDRT-PCR?
Which of the following actions utilize DDRT-PCR?
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DDRT-PCR can only identify genetic differences in cancer cells.
DDRT-PCR can only identify genetic differences in cancer cells.
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What small differences in sequence does DDRT-PCR allow researchers to detect?
What small differences in sequence does DDRT-PCR allow researchers to detect?
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What is the primary purpose of DDRT-PCR?
What is the primary purpose of DDRT-PCR?
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DDRT-PCR can only be performed on abundant mRNA samples.
DDRT-PCR can only be performed on abundant mRNA samples.
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What type of disorders has DDRT-PCR been used to gain insight into?
What type of disorders has DDRT-PCR been used to gain insight into?
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DDRT-PCR can help identify genes that are expressed only under certain __________ conditions in vivo.
DDRT-PCR can help identify genes that are expressed only under certain __________ conditions in vivo.
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Match the following effects with their corresponding applications of DDRT-PCR:
Match the following effects with their corresponding applications of DDRT-PCR:
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Study Notes
Differential Display
- A powerful technique for analyzing differences in gene expression
- Until 1992, subtractive hybridization was the only method to isolate differentially expressed genes
- In 1992, Liang and Pardee developed a new PCR-based technique called Differential Display (DD)
- DD focused on detecting differentially expressed genes among nearly 15,000 mRNA sequences in mammalian cells
- First described for comparing messages between normal and tumorigenic cells
- Effective identification and isolation of genes differentially expressed in various cells or under altered conditions is needed
Why Measure Gene Expression?
- More abundant genes/transcripts are assumed to be more important.
- Gene expression levels are assumed to correspond to protein levels.
- A normal cell has a standard expression profile/signature.
- Changes in the expression profile indicate events are occurring.
- Gene expression profiles represent a snapshot of cellular metabolism/activity at the molecular scale.
- They represent cumulative interactions of many hard-to-detect events or phenomena.
- Gene expression is a "proxy" measure for transcription/translation events.
Introduction
- The human genome has been completely sequenced.
- Of an estimated 30,000 genes in the human genome, perhaps only 10-15% are "turned on."
- Studying expressed genes has had a significant impact on biological research.
- The challenge has shifted from identifying the parts of the human genome to understanding their function in health and disease, a field called "functional genomics" or "post-genomic area."
High Throughput Measurement
- Genomics: DNA
- Transcriptomics: RNA
- Proteomics: Protein
- Metabolomics, Phenomics (etc.): Metabolite, Phenotype
- A chart depicts a visual representation from easier to more challenging aspects of measurement.
Measuring Gene Expression
- Hybridization: Northern/Southern Blotting, DNA Microarrays or Gene Chips
- Sequencing: Serial analysis of gene expression (SAGE), RNA-Sequencing
- Polymerase chain reaction (PCR)-based approaches: RT-PCR (real-time PCR), Differential display PCR (DD-PCR)
Northern Blotting
- Used to study gene expression by detecting RNA.
- Also known as the RNA blot.
- Workflow:
- Separates mRNA on an agarose gel.
- Transfers mRNA to a nitrocellulose filter.
- Denatures and hybridizes with 32P-labeled complementary DNA probe.
- Probe binds to membrane and forms double-stranded DNA-RNA hybrid.
- Intensity of band indicates gene expression levels.
Advantages and Disadvantages of Northern Blotting
- Advantages: Inexpensive, quantitative method for measuring transcript abundance; well-used and well-understood technology; sensitive due to radioactive probes.
- Disadvantages: Relies on radioactive labeling ("dirty" technology); quality control issues; old-fashioned technology; largely replaced by microarrays and other technologies.
Microarray
- High-throughput technology using Cy3 and Cy5 fluorescence for detection.
- Measures the gene expression profile of a cell.
- Microarrays are hybridized with labeled cDNA synthesized from a mRNA sample of some tissue
- Intensity of label (radioactive or fluorescent) on each spot indicates gene expression.
- One-dye arrays show the absolute expression level of each gene.
- Two-dye arrays show the relative expression level of the same gene in two samples (labeled with different colors and mixed before hybridization).
SAGE (Serial Analysis of Gene Expression)
- SAGE = Serial Analysis of Gene Expression
- Converts every mRNA molecule into a short (10-14 base), unique tag.
- Based on serial sequencing of 10-14 bp tags unique to each gene.
- Determines the absolute abundance of every transcript in a population of cells.
- Does not require a preexisting clone (unlike microarrays), allowing identification and quantitation of new genes/transcripts.
Advantages and Disadvantages of SAGE
- Advantages: Very direct and quantitative method of measuring transcript abundance; open-ended technology; built-in quality control.
- Disadvantages: Expensive and time-consuming; requires sequencing >50,000 tags per sample; best used with fully sequenced genomes; 3' ends of some genes can be highly polymorphic.
RNA-Seq
- Same concept as sequencing ESTs and counting SAGE tags.
- Describes a collection of experimental and computational methods to determine the identity and abundance of RNA sequences in biological samples.
- Sequences the cDNA from the mRNA component of a sample and compares against the transcriptome of another sample (using NGS).
Advantages and Disadvantages of RNA-Seq
- Advantages: High throughput; avoids radioactivity; kit systems and commercial suppliers make microarrays easy to use; uses many "high-tech" techniques and devices.
- Disadvantages: Relatively expensive; microarrays are subject to cross-hybridization bias; quality and quality control is highly variable; analysis and interpretation is difficult; measurements relative to a control specimen.
Reverse Transcriptase PCR
- Two kinds of "RT-PCR":
- One uses reverse transcriptase (RT) to produce cDNA from mRNA.
- Other uses real-time (RT) methods to monitor PCR amplification.
- Real-Time PCR measures and quantifies reaction during amplification.
- A quantitative method to quantify mRNA and cDNA in real-time.
- Measures fluorescence increase with each PCR cycle.
- Generates quantitative fluorescence data at the earliest PCR cycle phases (highest replication fidelity).
Advantages and Disadvantages of Real Time PCR
- Advantages:
- Sensitive assay
- Highly quantitative
- Highly reproducible
- Detects only a few molecules.
- Excellent dynamic range, linear over several orders of magnitude.
- Disadvantages:
- Expensive (instruments > $ 150K, materials also expensive).
- Not a high throughput system (10's to 100's of genes, not 1000's).
- Can pick up RNA carryover or contaminating RNA, leading to false positives.
Differential Display
- A popular method for isolating novel genes in various biological systems. (e.g., carcinogenesis, hormone regulation, plant biology, neurobiology)
- Basic Idea: Run two RNA (cDNA) samples side-by-side on a gel; excise and sequence bands present in one lane but not the other.
- Trick: Reduce sample complexity by making cDNA with primers to target a specific, smaller subset of transcripts.
Differential display PCR (DDRT-PCR)
- Takes advantage of RT-PCR, cDNA cloning, and DNA sequencing gel electrophoresis.
- Also known as DDRT-PCR or DD-PCR.
- A researcher typically studies at least two samples, but more can be studied if the experiment suggests it.
Differential Display-PCR (DDRT-PCR)
- A variation of standard PCR that allows for amplification.
- Displays subsets of mRNAs from different cell types or tissues.
- Can be used for isolating genes of interest.
- General Strategy:
- Reverse transcription using an anchor primer.
- PCR using the anchor primer and an arbitrary primer.
- Separating the PCR product by electrophoresis and visualization.
Advantages of DDRT-PCR
- Sensitive technique.
- No special equipment needed.
- Rapid and simple assays; increased sensitivity and reproducibility; possibility to perform effective search with very small amounts of RNA; ability to compare different cell populations; no prior information about mRNA needed.
Disadvantages of DDRT-PCR
- High frequency of false positive results (>70%).
- Primer binding to nonspecific sequences.
- Not useful in single mode; insufficient to cover all genes expressed in tissue; Not easily automated or scaled-up; fairly expensive to get hundreds of different clones.
Potential Applications of DDRT-PCR
- Answering biological questions in mammalian and other nonfungal systems. (Many same biological enigmas exist in medical mycology).
- Adaptable to fungal systems. -Examples (applications):
- Cell Differentiation
- Cell Cycle and Life Stages.
- Cell Activation and Signaling
- Markers and Mutations
- Drug Resistance and Targets for Drugs
- Nutritional and Environmental Stress
- Low Abundance Samples and In Vivo-Expressed Genes
- Pathogenesis.
Effective Modifications for Differential Display
- Reamplification (clone screening, precloning, vector sequences protocols)
- Screening (large screens, use of cDNA instead of RNA, detection methods-Radioactivity, staining, fluorescence,Chemiluminescence(DIG-labeled, no radiation) )
Reamplification: Methodology
- Isolation of cDNA: Excise cDNA fragments from agarose gels after initial amplification; purify.
- Ligation into vectors: Ligate the purified cDNA into suitable cloning vectors.
- Transformation: Introduce the ligated vectors into competent bacterial cells (e.g., E. coli).
- Screening: Plate transformed cells on selective media; screen colonies using: colony PCR, hybridization, sequencing .
Reamplification: Precloning
- Refers to preparation steps taken before cloning fragments into vectors to ensure fragments are appropriate for subsequent insertion/amplification.
- Methodology:
- Purification: Purify cDNA products from DDRT-PCR removing residual primers, nucleotides, and enzymes.
- Restriction enzyme digestion: Digest cDNA with restriction enzymes to create compatible ends for ligation; End repair or A-tailing.
Reamplification: Vector Sequences Protocols
- Methodology:
- Selection of Vector: Choose a vector based on factors such as promoter type(constitutive/inducible), selection markers (e.g., antibiotic resistance), and compatibility with downstream applications (e.g., expression systems.)
- Ligation Protocol: Follow specific protocols for ligating cDNA into vectors, including molar ratios of insert to vector (typically 3:1); incubation with T4 DNA ligase under optimal conditions.
- Transformation Efficiency: Use competent cells with high transformation efficiency.
Screening: Methodology
- 96-well technologies: Increased throughput; different samples/conditions in each well in a 96-well plate.
- Hybridization filter arrays: Enables simultaneous hybridization of cDNA samples to a membrane containing probes; rapid method for confirming differential expression.
- Reverse Northern assays: Hybridize labeled cDNA to a membrane with immobilized RNA; quick way to assess gene expression levels.
Detection
- Radioactivity: [a-35S]dATP; intense labeling of small fragments; increased physical protection required.
- Staining (e.g., silver staining): Direct visualization; less sensitive; procedures difficult to control and results inconsistent.
- Fluorescence: Safety, stability; low cost; disposal; high throughput useful when screening many mRNA species .
- Chemiluminescence (e.g., digoxigenin (DIG)-labeled cDNA probes): Sensitive, no radioactivity; easy quantification and visualization; enhancement of result reliability.
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Description
Test your knowledge on various molecular biology techniques, particularly SAGE, RNA-seq, and Reverse Transcriptase PCR. This quiz covers advantages, disadvantages, and applications of these technologies. Perfect for students and enthusiasts in the field of genetics and molecular biology.
