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Questions and Answers
What is the primary purpose of recombinant DNA technology?
What is the primary purpose of recombinant DNA technology?
DNA sequencing allows us to determine the precise sequence of amino acids in proteins.
DNA sequencing allows us to determine the precise sequence of amino acids in proteins.
False
What is the function of primers in DNA sequencing?
What is the function of primers in DNA sequencing?
To initiate DNA synthesis.
DNA amplification is commonly performed using ______.
DNA amplification is commonly performed using ______.
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Match the following terminologies with their corresponding definitions:
Match the following terminologies with their corresponding definitions:
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Which of the following is a characteristic of transgenic plants?
Which of the following is a characteristic of transgenic plants?
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Transgenic animals are easier to generate than transgenic plants.
Transgenic animals are easier to generate than transgenic plants.
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What is the main purpose of using transgenic bacteria?
What is the main purpose of using transgenic bacteria?
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Transgenic organisms contain _____ DNA that has been genetically modified.
Transgenic organisms contain _____ DNA that has been genetically modified.
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Match the following transgenic organisms with their applications:
Match the following transgenic organisms with their applications:
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What is the primary purpose of gel electrophoresis in DNA sequencing?
What is the primary purpose of gel electrophoresis in DNA sequencing?
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DNA fragments move towards the negatively charged electrode during gel electrophoresis.
DNA fragments move towards the negatively charged electrode during gel electrophoresis.
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What charge do DNA strands carry that influences their movement in gel electrophoresis?
What charge do DNA strands carry that influences their movement in gel electrophoresis?
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In gel electrophoresis, smaller DNA fragments move ______ than larger fragments.
In gel electrophoresis, smaller DNA fragments move ______ than larger fragments.
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Match the following steps in the DNA sequencing process with their descriptions:
Match the following steps in the DNA sequencing process with their descriptions:
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What significant achievement is the Human Genome Project known for?
What significant achievement is the Human Genome Project known for?
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Restriction enzymes are used to join DNA fragments together.
Restriction enzymes are used to join DNA fragments together.
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What is the purpose of plasmids in recombinant DNA technology?
What is the purpose of plasmids in recombinant DNA technology?
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The __________ leaves single-stranded fragments that are complementary to each other.
The __________ leaves single-stranded fragments that are complementary to each other.
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Match the following components of recombinant DNA technology with their functions:
Match the following components of recombinant DNA technology with their functions:
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Study Notes
Lecture Outcomes
- Learn how recombinant DNA technology is used to study development and disease
- Understand DNA sequencing and gene cloning
- Familiarize yourself with DNA amplification by PCR
- Learn how genetic engineering is used to create transgenic animals and for gene therapy
Biotechnology
- The technical application of biological knowledge and engineering for human purposes
- Includes commercial, industrial, and health applications
- Includes recombinant DNA technology, genetic engineering, and gene therapy
Recombinant DNA Technology
- Recombinant DNA combines DNA from two or more sources
- DNA sequencing determines the precise sequence of bases in DNA
- Uses primers to initiate DNA synthesis
- Fluorescently labeled nucleotides are added one at a time during sequencing, and the process stops after each modified nucleotide.
- Enzymes facilitate nucleotide addition to the growing DNA strand
- DNA polymerase replicates DNA
- Gel electrophoresis separates DNA fragments
DNA Sequencing
- Step 1: Amplify DNA fragment using DNA polymerase and mix with primers and labeled nucleotides.
- Primers bind to a region in the DNA and act as a starting point for synthesis of new DNA strand
- Fluorescent nucleotides are added one-by-one, based on complementarity
- Facilitated by an enzyme (polymerase)
- Synthesis can be done with normal or modified nucleoids. Synthesis stops every time a modified nucleotide is added
- Step 2: DNA strands separated by size using gel electrophoresis. DNA, which is negatively charged, moves towards the positively charged electrode. Smaller bands move faster in the gel
- Step 3: Laser scans the gel, reading the locations of nucleotides; a graphic displays nucleotides in the new strands, arranged by size.
The Human Genome Project
- Mapped the human DNA sequence from 1990-2003
- Considered a major feat of the 20th century
- Optimized and used DNA sequencing technology
- Publicly available sequences of humans and other model organisms
Recombinant DNA Technology: Gene Cloning
- Recombinant DNA involves cutting, splicing, and copying DNA
- Restriction enzymes cut DNA at specific sites (often palindromes)
- DNA ligases join DNA fragments
- Plasmids assist in amplifying DNA by adding desired genes to a circular piece of bacterial DNA
Gene Cloning
- The same restriction enzymes are used because they leave single-stranded fragments that are complementary to other fragments.
- Fragments have complementary bases to match and bind to other fragments
- Researchers create circular bacterial plasmids containing human DNA
- The bacteria that contain the desired plasmids are selected and cloned
Cloning DNA Fragments: PCR
- Polymerase Chain Reaction
- Rapidly amplifies DNA sequences to obtain millions of copies
- Requires: DNA to be amplified, primers to bind to DNA template, and heat-stable DNA polymerase to synthesize new complementary DNA strands
- Repeated heating and cooling cycles allow for rapid amplification of defined DNA sequences.
PCR
- DNA is unwound by gentle heating
- Single strands of DNA are mixed with primers that bind to one end
- Nucleotides and DNA polymerase are added
- As the mixture cools, primers pair with ends of the template strands
- Nucleotides are attached in sequence and the DNA strands replicate, doubling the number of double stranded DNA molecules.
DNA Fingerprinting
- Identify the source of a DNA fragment
- Used in forensics, paternity testing, genealogy, and evolutionary studies
- Based on the principle that between genes, there are repeating sequences of base pairs called short tandem repeats (STRs)
- The number of times an STR repeats is highly variable between individuals.
- PCR is used to amplify and restriction enzymes to cut the target DNA.
- Fragments of varying sizes are separated and compared by gel electrophoresis
- Produces a pattern of separation: unique DNA fingerprint
Question break
- A question about identifying a suspect in a jewelry store robbery involving DNA evidence
- Suspects have different DNA profiles, displayed as patterns of separation, in a graphic showing blood and stains
Genetic engineering to create transgenic organisms
- Transgenic organisms are genetically modified to contain recombinant DNA
- Transgenic bacteria are used to manufacture things like insulin and growth hormones
- Transgenic plants may have increased resistance or more nutrients.
- Transgenic animals are more difficult to produce; it involves microinjection of foreign DNA into fertilized eggs
- Examples of successes include bovine growth hormone, which promotes faster animal growth, and transgenic mice, which study human diseases
Gene Therapy
- Introduce human genes into human cells to correct or treat diseases (like cystic fibrosis or hemophilia)
- Obstacles include introducing genes effectively, and assuring the genes are expressed where they are needed efficiently, preventing their passing to offspring.
Gene Therapy: Delivery of the desired gene to the patient cell.
- Gene Delivery is done by vectors or transporters
- Nonviral method includes liposomes, and electroporation
- Viral method includes retroviruses & adenoviruses
- FDA has approved a cell-based gene therapy for sickle-cell anemia; a new protein is introduced via lentivirus.
Review
- Recombinant DNA technology modifies DNA
- DNA sequencing identifies the order of nucleotides
- DNA fragments can be amplified using PCR
- Genes can be cloned using restriction enzymes
- STR testing uses restriction enzyme digestion and PCR
- Genetic engineering produces transgenic organisms
- Gene therapy treats human disease
Practice Questions:
- Questions on DNA sequencing, PCR, and gene pharming. (specific details below)
Practice Questions 1
- If DNA polymerase is faulty what is the result? (newly synthesized fragments will not migrate through the gel)
Practice Questions 2
- What is the most essential aspect of PCR? (primers matching the specific target region of the DNA)
Practice Questions 3
- How is spider silk generated in goat milk? (genes are isolated in spiders, placed in bacterial plasmids, inoculated into a goat's egg. The milk is analyzed and isolated to find the protein)
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Description
Test your knowledge on recombinant DNA technology, including the functions of primers, applications of transgenic organisms, and the principles of gel electrophoresis. This quiz covers essential concepts and terminologies in biotechnology. Challenge yourself and see how well you understand the advances in genetic engineering!
