Unit 10: Recombinant DNA, Cloning, and Biotechnology
The lab tools that cut, copy, read, and now directly edit DNA, and how each one turns a molecular trick into a working technology.
6 topics · 22 questions
Each topic is a path, not a menu: work down it in order and every question builds on the one above. Click any of them to watch the answer draw itself.
Restriction Enzymes and DNA Cloning
- How it worksHow does a restriction enzyme recognize a specific palindromic sequence and cut both DNA strands there?
- Worked exampleFind the sticky-end fragments produced when EcoRI cuts the sequence GAATTC on both strands.
- Going furtherWhat happens when a gene of interest cut with EcoRI is ligated into a plasmid also cut with EcoRI?
- Common mistakeWhy doesn't cutting a plasmid and a gene with the same restriction enzyme guarantee they will ligate together in the correct orientation?
Your turn1 of 2
A circular plasmid is cut by a restriction enzyme at 9 different recognition sites scattered around it. How many separate linear DNA fragments result?Gel Electrophoresis
- How it worksHow does an electric field pull negatively charged DNA fragments through a gel matrix by size?
- Worked exampleFind the order of DNA fragments of 200, 500, and 1500 base pairs from the wells to the bottom of a gel after electrophoresis.
- Common mistakeWhy doesn't gel electrophoresis separate DNA fragments by their base sequence instead of by their size?
Your turn1 of 2
A gel electrophoresis run separates DNA fragments of 200, 500, and 1500 base pairs, all loaded into the same well and run for the same amount of time. In what order do they end up, from closest to the wells to farthest down the gel?The Polymerase Chain Reaction
- How it worksHow do denaturation, annealing, and extension repeat each cycle to double the DNA amount in PCR?
- Worked exampleFind the number of DNA copies produced after 10 cycles of PCR starting from a single template molecule.
- Common mistakeWhy doesn't PCR need living cells the way cloning a gene into bacteria does?
- Going furtherWhat happens to a PCR reaction's specificity when the annealing temperature is set too low for the primers being used?
- Going furtherWhat happens to a PCR reaction's yield when the primers are designed to bind two nonspecific sites elsewhere in the genome?
Your turn1 of 2
Starting from a single template DNA molecule, how many copies exist after 19 cycles of PCR, given the amount of DNA doubles every cycle?DNA Sequencing: Sanger and Next-Generation Methods
- How it worksHow do dideoxynucleotides stop DNA synthesis at every possible position to build a ladder of fragments in Sanger sequencing?
- Going furtherWhat changes about sequencing speed and cost when massively parallel next-generation sequencing replaces the Sanger method?
- Ties togetherHow does DNA sequencing build on the same polymerase chemistry used in PCR and in normal cellular replication?
Your turn1 of 2
Sanger sequencing and PCR both rely on DNA polymerase extending a primer along a template strand, yet Sanger sequencing produces a ladder of different-length fragments instead of exponentially amplifying a full-length product. How does Sanger sequencing build on the same polymerase chemistry used in PCR and normal replication, while producing such a different result?CRISPR-Cas9 Gene Editing
- How it worksHow does a guide RNA direct the Cas9 enzyme to cut DNA at one specific target sequence?
- Worked exampleTrace how a cell's own double-strand break repair machinery either disables a gene or inserts a new sequence after a CRISPR cut.
- Common mistakeWhy doesn't CRISPR gene editing insert new DNA at every location in the genome that matches the guide RNA sequence?
- Going furtherWhat happens to an edited gene when a cell repairs a CRISPR cut using nonhomologous end joining instead of homologous recombination?
Your turn1 of 2
A guide RNA is designed to exactly match a 20-base target sequence in a genome. A student worries that CRISPR-Cas9 will insert new DNA at every location where that same 20-base sequence happens to occur by chance elsewhere in the genome. Why is that not automatically the case?Bacterial Transformation and Antibiotic Selection
- How it worksHow does plating transformed bacteria on antibiotic media select only the cells that took up the plasmid?
- Worked exampleTrace which bacterial colonies survive on ampicillin plates after a transformation where only 1 in 10,000 cells took up the plasmid.
- Common mistakeWhy doesn't every bacterial cell in a transformation take up a copy of the plasmid?
Your turn1 of 2
In a bacterial transformation, only 1 in 14232 cells takes up the plasmid carrying an ampicillin resistance gene. If 1368189 treated cells are plated on ampicillin-containing media, about how many colonies are expected to grow?