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Questions and Answers
What is the primary purpose of molecular cloning?
What is the primary purpose of molecular cloning?
Which of the following characteristics is NOT important when choosing a cloning vector?
Which of the following characteristics is NOT important when choosing a cloning vector?
What are the common vectors used in molecular cloning?
What are the common vectors used in molecular cloning?
What role does E. coli play in molecular cloning?
What role does E. coli play in molecular cloning?
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What is the purpose of using a selection marker in a cloning vector?
What is the purpose of using a selection marker in a cloning vector?
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What is the primary function of restriction endonucleases in recombinant DNA technology?
What is the primary function of restriction endonucleases in recombinant DNA technology?
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Which of the following describes the type of cuts made by a restriction enzyme that produces sticky ends?
Which of the following describes the type of cuts made by a restriction enzyme that produces sticky ends?
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What is the outcome of digesting DNA with restriction enzymes?
What is the outcome of digesting DNA with restriction enzymes?
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What feature of recognition sequences makes restriction endonucleases specific?
What feature of recognition sequences makes restriction endonucleases specific?
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How frequently does an 8 bp recognition sequence cut a random DNA sequence?
How frequently does an 8 bp recognition sequence cut a random DNA sequence?
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What is the role of DNA ligase in DNA manipulation?
What is the role of DNA ligase in DNA manipulation?
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What is a common application of restriction enzymes in recombinant DNA technology?
What is a common application of restriction enzymes in recombinant DNA technology?
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What problem do restriction endonucleases solve when working with foreign DNA?
What problem do restriction endonucleases solve when working with foreign DNA?
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What is the initial step in the PCR process?
What is the initial step in the PCR process?
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Which of the following is NOT a typical application of PCR?
Which of the following is NOT a typical application of PCR?
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What temperature is generally used to anneal the primers during PCR?
What temperature is generally used to anneal the primers during PCR?
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Which component is essential for the extension phase of PCR?
Which component is essential for the extension phase of PCR?
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How many cycles are typically repeated in the PCR process?
How many cycles are typically repeated in the PCR process?
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What is one use of PCR in the context of cancer treatment?
What is one use of PCR in the context of cancer treatment?
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Which method has PCR largely replaced due to its speed?
Which method has PCR largely replaced due to its speed?
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What specific information can PCR provide in forensic identification?
What specific information can PCR provide in forensic identification?
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What type of genetic alterations can Southern Blot detect?
What type of genetic alterations can Southern Blot detect?
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Which scenario might result in an aberrant sized mRNA that a Northern Blot could detect?
Which scenario might result in an aberrant sized mRNA that a Northern Blot could detect?
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What is a key limitation of Southern Blot?
What is a key limitation of Southern Blot?
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Which of the following statements about Polymerase Chain Reaction (PCR) is true?
Which of the following statements about Polymerase Chain Reaction (PCR) is true?
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What is a correct characteristic of Northern Blot technique?
What is a correct characteristic of Northern Blot technique?
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What does Southern Blot rely on for detecting genomic rearrangements?
What does Southern Blot rely on for detecting genomic rearrangements?
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What type of gene alteration would NOT be detected by a Southern Blot?
What type of gene alteration would NOT be detected by a Southern Blot?
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Why is the enzyme from Thermus aquaticus used in PCR?
Why is the enzyme from Thermus aquaticus used in PCR?
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What is the primary purpose of generating probes to detect homologous sequences?
What is the primary purpose of generating probes to detect homologous sequences?
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What differentiates cDNA libraries from genomic libraries?
What differentiates cDNA libraries from genomic libraries?
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Which of the following is NOT a reason for constructing a genomic library?
Which of the following is NOT a reason for constructing a genomic library?
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Why is the source of mRNA critical when constructing a cDNA library?
Why is the source of mRNA critical when constructing a cDNA library?
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What defines a genomic library in comparison to other types of libraries?
What defines a genomic library in comparison to other types of libraries?
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What is a key characteristic of cDNA synthesized using reverse-transcriptase?
What is a key characteristic of cDNA synthesized using reverse-transcriptase?
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What aspect of genomic libraries allows researchers to move along a chromosome?
What aspect of genomic libraries allows researchers to move along a chromosome?
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What is an important limitation of cDNA libraries compared to genomic libraries?
What is an important limitation of cDNA libraries compared to genomic libraries?
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What is the purpose of the neor gene in the creation of recombinant cells?
What is the purpose of the neor gene in the creation of recombinant cells?
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Which statement accurately describes the role of tkHSV in recombinant cells?
Which statement accurately describes the role of tkHSV in recombinant cells?
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What visual characteristic indicates chimeric mice in the gene-targeted knockout procedure?
What visual characteristic indicates chimeric mice in the gene-targeted knockout procedure?
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What is the primary method to identify black mice heterozygous for the black allele?
What is the primary method to identify black mice heterozygous for the black allele?
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What factor primarily influences the production efficiency of transgenic mice?
What factor primarily influences the production efficiency of transgenic mice?
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Which gene was introduced into the mouse genome to produce a larger animal?
Which gene was introduced into the mouse genome to produce a larger animal?
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What is the consequence of intercrossing black mice derived from ES cells?
What is the consequence of intercrossing black mice derived from ES cells?
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Which insertion type does not include tkHSV gene?
Which insertion type does not include tkHSV gene?
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Study Notes
Recombinant DNA Technology in Medicine
- Recombinant DNA technology involves techniques to manipulate DNA, including molecular cloning, DNA sequencing, Polymerase Chain Reaction (PCR), nucleic acid blotting (Southern and Northern analysis), protein production, and creation of knock-out, knock-in, and transgenic mice. Nucleic acid microarrays are also used to monitor gene expression levels in cells.
Restriction Endonucleases
- Restriction endonucleases are enzymes that cut DNA at specific, defined nucleotide sequences.
- They are purified from bacteria.
- These enzymes recognize short DNA sequences (usually 4-8 base pairs).
- Recognition sequences are palindromes, meaning the sequence is the same when read 5' to 3' on one strand and 3' to 5' on the other strand.
- Some enzymes create "sticky ends" (staggered cuts), while others create "blunt ends" (straight cuts).
- Restriction fragments are the DNA fragments produced by cleavage with a restriction enzyme.
- DNA ligase is used to covalently join the base-paired sticky ends.
Restriction-Recognition Sites
- Restriction-recognition sites are short DNA sequences that are recognized and cleaved by various restriction endonucleases.
- Specific examples are provided in tables about specific organisms and enzymes.
Selected Restriction Endonucleases and Their Restriction-Site Sequences
- Tables provide lists of restriction enzymes, their source microorganisms, recognition sites, and the types of ends produced (blunt or sticky). Examples of specific enzymes and their recognition sequences are listed
Restriction Fragments
- Restriction fragments are the pieces of DNA created when restriction enzymes cut DNA at specific sequences. Different lengths are produced depending on the sequence of the DNA and the restriction enzyme used.
Medical Uses of Gene Cloning
- Defining inherited mutations.
- Developing diagnostic tools.
- Providing prenatal testing and counseling.
- Determining the precise biochemical defect.
- Creating treatment therapies.
- Isolating functional genes with therapeutic potential.
- Protein production, without infectious risks from blood products
- Generating specific effects
- Designing vaccines
- Isolating genes with somatic mutations. (not inherited but developed within cells)
- Establishing diagnostics
- Measuring treatment response
- Targeting therapies to recognize altered genes.
Molecular Cloning
- Amplifying a specific DNA sequence or gene.
- This method differs from cloning organisms (replicating an entire organism from a single cell).
- Molecular cloning involves joining a DNA fragment to a vector (a DNA molecule capable of replication in a host cell, usually E. coli).
- Plasmids and bacterial viruses (retroviruses in eukaryotic cells) are common vectors for this purpose.
- Vectors are chosen for their size, ease of handling, and ability to generate many copies of the vector in a host organism, such as bacteria
- Vectors often have antibiotic resistance genes.
- Approximate maximum length of DNA that can be cloned using a particular vector type is provided in a table.
Plasmids
- Plasmids are extrachromosomal, double-stranded DNA molecules that replicate autonomously in bacterial cells.
- Plasmids provide a convenient method of cloning DNA, as they are small, easy to handle, and able to replicate
Cloning DNA Fragments
- Procedure of cloning a DNA fragment in a plasmid vector is detailed with diagrams.
Plasmid vectors
- Plasmids used for DNA cloning generally contain convenient restriction sites and a marker gene.
Simple Fusion of Fragments
- Procedures and diagrams detailing the simple fusion of different DNA fragments are shown.
pUC18/pUC19 Cloning Vectors
- Diagrams show the structure of pUC18 and pUC19 plasmids depicting the ampicillin resistance gene, lacZ gene, and polylinker region
Blue/White Selection
- A technique to identify colonies containing plasmids with inserted DNA fragments.
- Involves the use of IPTG and X-gal
- Positive selection: Colonies with intact lacZ gene break down X-gal, resulting in the development of a blue phenotype.
- Negative selection: Colonies with disrupted lacZ gene do not give a blue phenotype and appear white.
Some uses of cloning
- Isolating/characterizing a specific gene or mRNA.
- Creating probes to identify similar/homologous sequences (useful for diagnostics).
- Producing proteins on a large scale (therapeutic proteins).
- Generating mutations to form animal models of particular diseases.
DNA Libraries
- Collections of DNA fragments (from a genome or cDNA), each inserted into a vector.
- Genomic libraries contain DNA from the entire genome (coding regions, introns, regulatory elements).
- cDNA libraries contain DNA copies of mRNA (only the expressed portions of the genome).
cDNA Libraries
- cDNA libraries contain double-stranded DNA copies of mRNA sequences.
- cDNA libraries for a specific cell type only contain the sequences expressed in that cell.
- cDNA synthesis starts with reverse transcriptase
Construction of a Genomic DNA Library
- Procedure of constructing a genomic DNA library is explained.
Genomic Library
- Contains all the genetic information of an organism.
- Including introns, exons and noncoding regions.
- Useful for cloning the whole gene including regulatory sequences (5') for studying gene expression.
- Allows cloning of a new gene if the approximate chromosomal location is known.
- Enables creation of knock-out animal models.
cDNA Synthesis
- Procedure of generating a cDNA (copy DNA) is detailed.
Considerations for cDNA Libraries
- mRNA source is critical.
- No regulatory sequences.
Comparison of Genomic & cDNA Clones
- A comparison of genomic and cDNA clones is illustrated.
Nucleic Acid Hybridization
- Hybridization is a method for detecting specific nucleotide sequences in complex mixtures, such as those found in libraries.
- Based on base pairing of a single-stranded probe (known sequence) to a complementary sequence in the target. Probes are labeled so the hybrid can be detected (e.g., radioactive or fluorescent).
Membrane Hybridization Assay
- This method can be used to detect both DNA and RNA sequences.
Southern and Northern Blotting
- Procedures to separate nucleic acids by size and then probe them to detect specific sequences.
- Southern blotting: Analyzing DNA.
- Northern blotting: Analyzing RNA.
Southern Blot Applications
- Detecting large insertions and deletions in genes.
- Identifying copy number changes (amplification)
- Determining chromosomal rearrangements, such as translocations.
- Detecting mutations that create or destroy restriction enzyme sites
- Limitations: Not for point mutations
Northern Blot Applications
- Detecting changes in gene expression or RNA levels
- Measuring gene expression levels in different tissues or at different time points
- Identifying size variations in the mRNA transcript (insertions or deletions).
Polymerase Chain Reaction (PCR)
- A method to amplify a specific DNA sequence without cloning into a vector in clinical and forensic settings.
Uses primers flanking a target DNA region
- Short DNA sequences.
- Annealed to target DNA, forming new copies.
- Using high temperature-tolerant thermophilic enzymes (Taq polymerase)
- DNA amplified exponentially.
- Steps: Denature, Anneal specific primers, extend by polymerase, repeat cycles.
- Copied sequence can reach millions in just a few cycles.
PCR Continued
- Very sensitive test for detecting low DNA levels or RNA genome (for viruses or crime scenes).
- Faster than southern or northern blotting
PCR Technique
- Detailed visual steps of visual illustration of the PCR technique.
PCR Steps
- Denature the template DNA to separate strands.
- Anneal specific primers to single strands.
- Extend the strands using polymerase.
- Repeat to amplify.
Uses of PCR
- Detecting mutations.
- Diagnosing diseases.
- Determining the gender of embryonic cells.
- In forensic analysis (e.g., DNA fingerprinting).
- Gene cloning, carrier screening, screening for newborn.
Creating a Knockout Animal
- Procedure for generating knock-out mice (model of human diseases)
- Introduce DNA into ES cells with targeted disruption, causing mutation.
- Select cells with mutation using selective media.
- Transplant ES cells into embryos.
Cloning in Mice
- Using a target gene (e.g., human growth hormone), to introduce the gene into the mouse genome
- Demonstrating consequences of increased (unusual) gene expression
Types of DNA Polymorphism
- RFLP (Restriction Fragment Length Polymorphism).
- VNTR (Variable Number of Tandem Repeats).
- STR (Short Tandem Repeats).
- SNP (Single Nucleotide Polymorphism)
Sanger (Dideoxy) Sequencing
- Method to determine the DNA sequence.
- Uses dideoxynucleotides (dideoxynucleotides (ddNTPs) which terminate the DNA chain, leading to various lengths of the product) and labeled ddNTPs, with a labeled primer
- Separated by electrophoresis.
- The sequence of colors can be read from the results to determine the DNA sequence.
Automated DNA Sequencing
- Using fluorescently labeled dideoxynucleotides.
- Separated by electrophoresis using a capillary tube.
- Sequence read directly from the computer.
Production of High Levels of Proteins from Cloned cDNA
- Desired cDNA is cloned.
- Synthesized in engineered E. coli cells in large quantities.
- Expression vectors are used (e.g., using promoter systems).
E. coli Expression Vector
- A plasmid vector where the target gene is placed to create a functional expression vector. The gene can be translated to produce the protein of interest in engineered E. coli cells.
Other Expression Systems
- Two-step DNA expression systems (using T7 RNA polymerase and T7 promoter) for high-level protein expression in bacteria.
Recombinant DNA Products in Medicine
- Tabular summary of some recombinant DNA products including examples of their use.
Key Terms
- The terms used in this summary with descriptions.
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Description
This quiz explores the fundamentals of recombinant DNA technology, including techniques such as molecular cloning and PCR. It also covers the role of restriction endonucleases in DNA manipulation and gene expression monitoring. Test your understanding of these essential concepts in modern biotechnology.
