Podcast
Questions and Answers
What is the primary purpose of restriction enzymes in recombinant DNA technology?
What is the primary purpose of restriction enzymes in recombinant DNA technology?
Which of the following describes the nature of most recognition sequences of restriction enzymes?
Which of the following describes the nature of most recognition sequences of restriction enzymes?
What is targeted mutagenesis primarily used for in molecular genetics?
What is targeted mutagenesis primarily used for in molecular genetics?
How do engineered nucleases differ from natural restriction enzymes?
How do engineered nucleases differ from natural restriction enzymes?
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What characteristic feature do sticky ends produced by restriction enzymes have?
What characteristic feature do sticky ends produced by restriction enzymes have?
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What role do CRISPR-Cas systems play in molecular genetics?
What role do CRISPR-Cas systems play in molecular genetics?
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Which of the following techniques is specifically used for copying DNA fragments?
Which of the following techniques is specifically used for copying DNA fragments?
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What is a key advantage of using recombinant DNA technology?
What is a key advantage of using recombinant DNA technology?
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Study Notes
Molecular Genetic Techniques to Understand Species Biology
- Molecular genetic techniques are used to study species biology.
- Students will learn about techniques to cut and visualize DNA molecules.
- Students will learn how to copy DNA fragments with PCR.
- Methods for determining the base sequence of a DNA fragment will be discussed.
- Techniques for analyzing gene function, including targeted mutagenesis, will be covered.
- Practical applications of molecular genetics will be described.
Recombinant DNA Technology
- Recombinant DNA technology is a set of molecular techniques for locating, isolating, altering, and studying DNA segments.
- It frequently combines DNA from two distinct sources.
- Examples include combining genes from two different bacteria or inserting a human gene into a viral chromosome.
- This process is often called genetic engineering.
Restriction Enzymes
- Restriction enzymes are naturally produced by bacteria to defend against viruses.
- Bacteria protect their own DNA by modifying the recognition sequence, usually by adding methyl groups to the DNA.
- Over 800 different restriction enzymes recognize and cut DNA at more than 100 different sequences.
Engineered Nucleases
- Engineered nucleases are used to overcome the limitations of restriction enzymes.
- These consist of a portion of a restriction enzyme that cuts nonspecifically coupled with another protein that recognizes a specific DNA sequence.
- Zinc-finger nucleases (ZFNs) use a DNA binding domain called a zinc finger attached to a restriction enzyme.
- Transcription activator-like effector nucleases (TALENs) use a protein that normally binds to sequences in promoters attached to the FokI restriction enzyme.
Genome Editing with CRISPR-Cas
- CRISPR-Cas systems are naturally found in bacteria and archaea.
- These systems protect the organisms against bacteriophages, plasmids, and other invading DNA elements.
- CRISPR RNAs (crRNAs) are encoded by DNA sequences called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).
- CRISPR arrays consist of palindromic sequences separated by unique spacers derived from bacteriophages or foreign plasmids.
Separating and Viewing DNA Fragments
- Gel electrophoresis separates DNA molecules based on their size and electrical charge.
- DNA samples are placed in wells in an agarose gel.
- An electrical current passes through the gel, causing DNA fragments to migrate.
- Smaller fragments migrate faster than larger fragments.
- A dye specific for nucleic acids is added to visualize the DNA fragments as bands on the gel.
Locating DNA Fragments with Probes
- Probes are DNA or RNA with complementary sequences to a gene of interest.
- Probes pair with complementary sequences in a process called hybridization.
- Southern blotting is used to transfer denatured DNA fragments to a solid medium (like nylon membrane) for hybridization with a labeled probe.
- The labeled probe identifies specific DNA fragments.
PCR (Polymerase Chain Reaction)
- PCR is an enzymatic, in vitro method used to amplify DNA fragments rapidly.
- DNA is heated to separate the two strands.
- Short primers attach to the target DNA.
- DNA polymerase synthesizes new DNA strands from the primers.
- Each cycle of PCR doubles the amount of DNA.
Crucial Innovations in PCR
- The discovery of Taq polymerase, a heat-stable DNA polymerase from Thermus aquaticus, made it possible for PCR to be repeated with the heat denaturation step.
- Automated thermal cyclers allow the rapid change of temperatures needed for PCR's multiple cycles.
Limitations of PCR
- PCR requires prior knowledge of at least part of the target DNA sequence for primer design.
- The capacity of PCR for amplifying extremely small DNA amounts makes contamination a significant problem.
- Taq polymerase does not proofread, leading to occasional errors in the copied sequence.
- The size of fragments amplified using standard Taq polymerase is typically less than 2000 bp.
DNA Sequencing
- DNA sequencing is a powerful method for analyzing DNA sequences.
- Dideoxy sequencing, developed by Sanger, relies on the elongation of DNA by DNA polymerase and incorporation of dideoxyribonucleotides (ddNTPs) that terminate DNA synthesis at specific bases creating fragments.
- Next-generation sequencing is quicker and less expensive than Sanger sequencing.
- DNA fingerprinting uses repeated sequences in DNA (like STRs) to distinguish individuals.
Next-Generation Sequencing Technologies
- Illumina sequencing is the most common next-generation sequencing method.
- It sequences many DNA fragments simultaneously.
- Third-generation sequencing is a newer sequencing method that does not require a pause between the addition of nucleotides. Examples include BioPac third-generation sequencing.
Analyzing Gene Function
- Forward genetics starts with a phenotype and works backward to identify the gene or genes responsible.
- reverse genetics starts with a gene sequence and works forward to discover its function and effect on the phenotype.
- Creating random mutations: introducing changes in the DNA sequence can be induced by environmental factors like radiation or chemical mutagens.
- Targeted mutagenesis: allows mutations at specific DNA locations, like using CRISPR-Cas9. This is useful when appropriate restriction sites are not available.
- Transgenic animals: introducing a gene to an organism that normally lacks it to observe the effects on the phenotype, providing a way to study gene function.
- Knockout mice: a mouse that has a particular gene disabled by inserting foreign DNA sequences, which can reveal the gene's function.
- Silencing genes with RNA interference (RNAi): uses small RNA molecules called siRNAs to temporarily turn off gene expression. This allows researchers to study the effects of gene absence and helps identify genes involved in normal organism function.
Biotechnology Applications
- Molecular genetic techniques are used in pharmaceuticals, specialized bacteria, agriculture, genetic testing, and gene therapy.
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