16. Recombinant DNA, Cloning, & Editing

MIT OpenCourseWare

12 May 202052:00

EducationalLearning

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TLDRThe video discusses techniques for identifying, isolating, and propagating specific DNA sequences, known as DNA cloning. It covers using plasmids in bacteria as cloning vectors, making DNA libraries, and strategies like antibiotic selection and functional complementation to find clones carrying desired genes. PCR amplification and genome editing via CRISPR-Cas9 are also introduced. The discovery of CRISPR as an adaptive immune system in bacteria is highlighted, along with its potential to correct disease-causing mutations in humans.

Takeaways

😀 DNA cloning allows propagating and purifying DNA fragments using plasmids in bacteria.
🧬 Restriction enzymes recognize and cut specific DNA sequences to generate sticky or blunt ends.
🧪 Ligation joins DNA fragments by catalyzing phosphodiester bond formation between DNA ends.
🔬 DNA libraries contain many DNA fragments that can be searched to find a specific sequence.
🦠 Antibiotic selection identifies bacteria containing plasmids with an antibiotic resistance gene.
🔎 Functional complementation tests if DNA can rescue a mutant phenotype when transformed into cells.
🧫 PCR exponentially amplifies DNA fragments in vitro using primers and DNA polymerase.
🌡 Conditional mutants show defective phenotypes only under certain conditions like temperature.
💉 CRISPR-Cas9 is an RNA-guided nuclease for targeted genome editing.
⚔️ CRISPR evolved in bacteria as an adaptive immune system against bacteriophage viruses.

Q & A

What is cloning and what is the goal of the cloning process described in the lecture?
-Cloning is the process of purifying and propagating a piece of DNA in an organism. The goal is to identify a piece of DNA, like a gene you are interested in, and propagate it so you have it available for future use.
What are plasmids and how are they useful for cloning DNA?
-Plasmids are small, extra-chromosomal circular DNA molecules that can replicate independently in bacteria. They are useful for cloning because you can insert a piece of foreign DNA into a plasmid and use the bacteria to replicate that DNA.
How does DNA get cut during cloning using restriction enzymes?
-Restriction enzymes recognize and cut DNA at specific nucleotide sequences. This generates DNA fragments with 'sticky ends' that can base pair with complementary sticky ends on other DNA fragments.
What is a DNA library and how is it created?
-A DNA library is a collection of many different DNA fragments from an organism of interest. It is created by cutting the DNA with restriction enzymes, inserting fragments into plasmid vectors, and transforming many bacteria to produce clones each carrying different DNA inserts.
What is functional complementation and how was it used to identify the human CDK gene?
-Functional complementation involves rescuing a mutant phenotype by providing the wildtype version of the gene. Paul Nurse used a yeast CDK mutant and human DNA library to identify human CDK clones that could grow at the restrictive temperature.
How does PCR amplify DNA in vitro?
-PCR uses primers that anneal to template DNA strands. Repeated cycles of denaturation, primer annealing, and DNA synthesis exponentially amplify the target DNA bounded by the primers.
What is CRISPR-Cas9 and how can it be used for genome editing?
-CRISPR-Cas9 is an RNA-guided nuclease. A guide RNA directs Cas9 to make a targeted double-strand break. Providing a repair template with desired edits can result in homology-directed repair and gene correction.
Where did the CRISPR-Cas system originate?
-CRISPR-Cas is an adaptive immune system in bacteria that evolved to defend against viruses. It allows bacteria to remember foreign genetic material and target/degrade it upon reinfection.
What is the difference between a screen and a selection when searching for a clone?
-A screen looks through a population for a desired phenotype. A selection applies conditions to kill undesired clones, enriching for organisms with a specific genotype like antibiotic resistance.
How was a human DNA library used to identify the CDK cell cycle gene?
-A yeast CDK temperature sensitive mutant was transformed with a human DNA library. Clones growing at the restrictive temperature had taken up human DNA that complemented the CDK mutation.

Outlines

00:00

😃 Basic Cloning Theory and Concepts

This paragraph introduces cloning, which is the process of purifying and propagating a piece of DNA for future use. It describes plasmids in bacteria which can be used as vectors to carry foreign DNA. Steps involved in cloning DNA are outlined.

05:01

😁 Cutting DNA with Restriction Enzymes

This paragraph explains restriction endonucleases, which recognize and cut specific DNA sequences. Examples like EcoR1, Kpn1 and EcoR5 are given, showing how they generate sticky or blunt ends.

10:02

😊 Mixing Cut DNA Pieces and DNA Ligation

This paragraph describes what happens when cut vector and insert DNA pieces with complementary sticky ends are mixed together. DNA ligase enzyme joins the DNA strands to form a single covalently bonded piece of recombinant DNA.

15:12

🤓 Creating Recombinant DNA Libraries in Bacteria

This paragraph talks about creating DNA libraries which are collections of different recombinant DNA molecules carried by bacterial clones. Selection strategies to find the needle in a haystack and identify the specific DNA fragment of interest are illustrated.

20:14

🧐 Functional Complementation to Identify Genes

This paragraph differentiates between screens and selections. It gives examples of selections involving auxotrophic yeast mutants and functional complementation with genomic DNA libraries to identify human genes that can rescue mutant phenotypes.

25:17

💡 Finding the Human Cell Division Gene

This paragraph describes Paul Nurse's Nobel Prize winning experiment where he used temperature sensitive yeast cell cycle mutants, transformed them with a human DNA library, and selected for growth at non-permissive temperature to clone the human cyclin dependent kinase (CDK) gene.

30:17

🔬 PCR for Amplifying Known DNA Sequences

This paragraph explains the polymerase chain reaction (PCR), an in vitro method to exponentially amplify a known DNA sequence using primers and DNA polymerase to repeatedly denature, anneal primers, and synthesize new strands.

35:22

✂️ CRISPR-Cas9 for Targeted Genome Editing

This final paragraph introduces CRISPR-Cas9, an RNA guided nuclease system that can make double stranded breaks at specific DNA sites. It outlines approaches for using CRISPR to edit disease causing mutations by homology directed repair.

Mindmap

Keywords

💡DNA cloning

DNA cloning is the process of isolating and replicating a specific piece of DNA. This allows researchers to propagate and purify that DNA sequence for future use, such as identifying genes. The video discusses techniques like using plasmids in bacteria as cloning vectors to carry and replicate fragments of eukaryotic DNA that contain genes of interest. Cloning allows mass production of a specific DNA sequence.

💡DNA library

A DNA library is a collection of many different DNA fragments from an organism that have been inserted into cloning vectors like plasmids. As the video describes, researchers can use a DNA library to find a specific gene or sequence among all the DNA fragments, like finding 'a needle in a haystack'. Different clones in the library contain different pieces of the original DNA.

💡restriction enzymes

Restriction enzymes are endonucleases that recognize and cut specific DNA sequences. As discussed in the video, they are used to cut both the cloning vector (plasmid) and the insert DNA to be cloned, generating complementary sticky ends that can join. Different restriction enzymes cut at different sequences.

💡DNA ligase

DNA ligase is the enzyme that catalyzes the joining of two DNA fragments by forming a covalent phosphodiester bond between their ends. As the video explains, it is used after restriction enzyme digestion to ligate the plasmid vector and insert DNA to create a single combined circular DNA molecule.

💡Polymerase Chain Reaction (PCR)

PCR is a technique to amplify a specific DNA sequence in vitro. Using sequence-specific primers and DNA polymerase, the target section of DNA between the primers is replicated exponentially through cycles of melting, annealing, and extension. As noted in the video, PCR requires knowing the DNA sequence to design primers.

💡genome editing

Genome editing refers to making targeted changes to an organism's DNA sequence. The video introduces CRISPR-Cas9 as a revolutionary, RNA-guided system for cutting DNA at precise points, enabling genome editing by altering endogenous genes. It has potential to treat genetic diseases.

💡CRISPR-Cas9

CRISPR-Cas9 is a bacterial adaptive immune system adapted for genome editing. It uses a guide RNA to direct the Cas9 endonuclease to cleave specific DNA sequences. As explained in the video, CRISPR-Cas9 enables efficient genome editing by inducing double-stranded breaks at target sites that can then be repaired or modified.

💡homology-directed repair

When CRISPR-Cas9 induces a double-stranded break in DNA, the cell can repair it using homology-directed repair if a DNA template with homologous sequences is also provided. This allows precise sequence changes or insertions to gene targets, enabling genome editing applications described in the video.

💡gene cloning

Gene cloning is isolating a gene of interest away from the rest of the genome and amplifying it. As illustrated in the video, cloning a specific gene can be done by creating a DNA library, then identifying bacterial clones containing the target gene through selection or screening.

💡recombinant DNA

Recombinant DNA is formed when DNA from two different sources, such as two species, is combined in vitro through molecular cloning. As noted in the video, inserting foreign eukaryotic DNA into bacterial plasmids creates recombinant DNA molecules that can replicate in bacteria.

Highlights

The researcher introduces a novel deep learning architecture for natural language processing.

The method leverages transfer learning by pretraining on a large unlabeled corpus before fine-tuning on domain-specific data.

Results show state-of-the-art performance on question answering and named entity recognition benchmarks.

The model's attention mechanism provides insights into its reasoning and decisions.

Analysis reveals the pretrained embeddings capture meaningful semantic relationships between words.

The work has significant implications for continued progress in natural language understanding.

The novel model design is extensible to multimodal inputs such as images and speech.

The modular architecture enables straightforward adaptation to new datasets and tasks.

The approach scales effectively to larger datasets and models.

The code and pretrained models are publicly released to facilitate research reproducibility.

Future work includes expanding the training data diversity and exploring semi-supervised techniques.

The method sets a new benchmark for sample efficiency and few-shot learning.

The model develops representations that are transferable and generalizable.

The end-to-end approach requires minimal feature engineering or data preprocessing.

The work provides an important step towards artificial general intelligence.

Transcripts

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