Ch. 9: Biotechnology

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

Which type of colonies indicate the presence of a recombinant plasmid in blue-white screening?

White colonies (correct)

Blue colonies

Yellow colonies

Green colonies

What is the role of restriction enzymes in producing human insulin in bacteria?

To synthesize insulin directly in bacteria

To transform bacteria with foreign DNA

To enhance the growth of bacteria

To cut DNA and plasmids for gene insertion (correct)

What happens to bacteria without recombinant plasmids when grown on X-gal media?

They produce white colonies

They transform into a mutant form

They produce blue colonies (correct)

They cannot grow at all

Which of the following describes the purpose of blue-white screening?

To identify and select for bacteria with recombinant plasmids Signup and view all the answers

In the context of insulin production for Type-I Diabetes, what is the main challenge addressed?

Insufficient insulin production by the pancreas Signup and view all the answers

What characterizes a recombinant plasmid?

It contains an inserted gene of interest Signup and view all the answers

What is the primary function of restriction enzymes in genetic engineering?

To cut DNA at specific recognition sites. Signup and view all the answers

Which characteristic is true about plasmids?

They exist as circular rings of double-stranded DNA. Signup and view all the answers

What defines a vector in genetic engineering?

A vehicle that transfers DNA and self-replicates. Signup and view all the answers

What is a viral vector?

An inactive virus used for gene transfer. Signup and view all the answers

How do restriction enzymes create 'sticky ends'?

By cutting both strands at specific sequences to create overhanging ends. Signup and view all the answers

What is the purpose of using plasmids in recombinant DNA technology?

To insert foreign genes into bacteria for replication. Signup and view all the answers

What happens when a restriction enzyme cuts DNA?

It generates complementary overhanging ends. Signup and view all the answers

Which method is commonly used in biotechnology to transfer genes into bacteria?

Utilizing transformation or conjugation. Signup and view all the answers

What is a characteristic of shuttle vectors?

They can exist in multiple host organisms for gene cloning. Signup and view all the answers

What is the primary purpose of PCR in biotechnology?

To rapidly copy a DNA sample Signup and view all the answers

Which component is essential for the annealing step in PCR?

Primers Signup and view all the answers

What property of Thermus aquaticus's DNA polymerase makes it suitable for PCR?

It can withstand high temperatures. Signup and view all the answers

During which step of PCR is the DNA double helix separated?

Denaturation Signup and view all the answers

What is formed from mRNA in reverse-transcription PCR?

Complementary DNA (cDNA) Signup and view all the answers

How many times does the DNA quantity double in PCR after each round of copying?

Once Signup and view all the answers

Which of the following is NOT one of the four essential items needed to perform PCR?

RNA polymerase Signup and view all the answers

What are nucleotides in the context of PCR?

Building blocks of DNA that are added during amplification Signup and view all the answers

Which of the following describes the overall objective of biotechnology?

To modify living organisms for beneficial purposes Signup and view all the answers

What is the role of salt and buffers in PCR?

To stabilize DNA and primers Signup and view all the answers

What happens to DNA during the extension step of PCR?

DNA polymerase extends the primer to copy the DNA. Signup and view all the answers

What is the significance of cooling DNA to 60 ⁰C in PCR?

It facilitates the natural annealing of primers. Signup and view all the answers

What is the process by which DNA quantity increases in PCR?

It doubles each cycle. Signup and view all the answers

Which temperature is optimal for DNA polymerase activity in PCR?

72 ⁰C Signup and view all the answers

Which of the following components is NOT involved in polymerase chain reaction (PCR)?

Restriction enzymes Signup and view all the answers

What type of DNA synthesis involves building genes using a synthesis machine?

Synthetic DNA synthesis Signup and view all the answers

Which method allows for fast and cheap sequencing using synthetic labeled nucleotides?

Next Generation Sequencing Signup and view all the answers

Which step is not part of the PCR cycle?

Translation Signup and view all the answers

What is the main function of vectors in genetic engineering?

To transfer genes into cells Signup and view all the answers

What is the outcome of a successful PCR process after repeating all steps numerous times?

An exponential increase in DNA quantity Signup and view all the answers

What is the primary function of a viral vector in gene transfer?

To deliver DNA into host cells without producing new virus Signup and view all the answers

Which method involves removing cell walls from bacteria to facilitate gene transfer?

Protoplast Fusion Signup and view all the answers

What does electroporation achieve in the context of DNA transfer?

It opens cell membrane pores using an electrical pulse Signup and view all the answers

Which gene transfer method uses a device that shoots DNA-coated particles into cells?

Gene gun Signup and view all the answers

What is one limitation of viruses regarding replication?

They require a host cell to replicate Signup and view all the answers

In the context of biotechnology, what does 'transformation' refer to?

The uptake of DNA by cells from their environment Signup and view all the answers

What occurs to viral DNA after it is incorporated into a host's genome by a viral vector?

It transcribes and translates using the host’s cellular machinery Signup and view all the answers

Which method for transferring DNA directly injects genetic material into a cell?

Microinjection Signup and view all the answers

Which of these is NOT a way to transfer DNA into cells?

Genomic sequencing Signup and view all the answers

What is created when a gene of interest is spliced into the viral DNA of a viral vector?

A modified viral vector for gene transfer Signup and view all the answers

Study Notes

Restriction Enzymes

Cut DNA at specific recognition sites
Cut both DNA strands
Create "sticky ends" which are overhanging ends that are complementary to each other
Restriction enzymes can create "sticky ends" in both source and vector DNA. This creates complimentary ends.
The same restriction enzymes can be used to cut both source and vector DNA.

Recombinant DNA Technology

Uses restriction enzymes to create recombinant plasmids.
Recombinant DNA plasmids are created by inserting a gene from one organism into another via a plasmid.
Plasmids can move into and out of bacterial cells.
Plasmids replicate, transcribe, and translate independently of the bacterial chromosome.

Vectors

"Vehicles" used to transfer DNA from a source to a target cell.
Vectors must be able to self-replicate.
Shuttle vectors exist in different species and can move cloned sequences among various organisms.
Two common types of vectors are plasmids and viral vectors.

Plasmids

Natural "free-floating" circular DNA.
Double stranded DNA.
Replicated, transcribed, and translated once in bacteria.
Can be inserted into bacterial cells.
Resistance plasmids provide resistance to certain antibiotics.

Viral Vectors

"Disabled" viruses used to infect cells and transfer DNA.
Unable to reproduce but can still infect cells.
Viral vectors are inserted into cells by spliced into the viral DNA.
The DNA is then incorporated into the host genome and transcribed and translated by the host cell.

5 Additional Ways to Transfer DNA into Cells

Transformation: Cells take up DNA from the surrounding environment.
Protoplast Fusion: Removing cell walls from two bacteria allows them to fuse.
Electroporation: Introducing DNA into bacteria or other cells using electrical pulse to briefly open the pores in the cell membranes.
Gene Gun: Uses elemental particles of a heavy metal coated with DNA to deliver exogenous DNA to cells.
Microinjection: Uses a glass micropipette to inject foreign DNA into a cell.

Polymerase Chain Reaction (PCR)

Rapidly copies DNA samples.
Results in millions or billions of DNA copies in 1 hour.
Used for creating millions of gene copies for:
- Transgenic and forensic biology.
- Diagnostic tests for genetic diseases.
- Detecting pathogens.
- Studying gene function.
Reverse-transcription PCR uses mRNA as a template and a reverse transcriptase enzyme.
Creates cDNA (complimentary DNA) - a complimentary DNA strand from an mRNA sequence.

PCR Requirements

DNA: The DNA to be amplified.
Primer: A short DNA sequence complementary to the beginning of a DNA sequence of interest.
DNA Polymerase: An enzyme that adds nucleotides.
Nucleotides: dtp's: dtpA, dtpT, dtpC and dtpG (dtp = deoxyribose tri-phosphate)
Salt and buffers.

PCR Steps

Denature DNA: Heat DNA to 95⁰C to separate double stranded DNA.
Anneal Primers: Cool DNA to 60 ⁰C, primers will anneal to the DNA sequence by base pairing.
Extension of DNA: Heat DNA to 72 ⁰C, DNA polymerase extends the primer, creating a copy of the DNA.

Synthetic DNA

Builds genes using a DNA synthesis machine.
Nucleotides, primers, and genes can be easily synthesized.
Synthetically labeled nucleotides are used in next-generation sequencing, which is a fast and cheap method of sequencing.

Blue-White Screening

A method for selecting recombinant plasmids.
White colonies: Recombinant DNA colonies, the plasmid contains an inserted gene.
Blue colonies: Non-recombinant cells, the plasmid does not contain an inserted gene.

Human Uses for Transgenic Biology

Gene Therapy: Replacing defective or missing genes.
Transgenic Bacteria: Growing human enzymes and other proteins in bacteria, such as insulin.

Producing Human Insulin in Bacteria

Bacteria can be used to produce human insulin.
Type 1 diabetes is caused by the pancreas not making enough or any functional insulin.
Insulin is a protein hormone that signals cells to bring glucose into the blood.
Transgenic insulin procedure:
1. Restriction enzymes are used to cut the human insulin gene out of the donor cell and also to cut the plasmid.
2. The gene is then inserted into the plasmid.
3. The modified plasmid is then introduced into bacteria.
4. The bacteria will produce the human insulin protein.

Gene Silencing

Gene silencing is a process that can be used to block the expression of a specific gene.
Gene silencing has been used to develop new treatments for various diseases, including cancer.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

This quiz covers key concepts in Recombinant DNA Technology, emphasizing the role of restriction enzymes, plasmids, and vectors. Understand how these elements work together to create recombinant DNA plasmids and their applications in genetic engineering.