Molecular Biology: Reverse Transcriptase and Vectors

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What is the maximum insert size that can be accommodated by a pBAC?

500 kbp

100 kbp

50 kbp

300 kbp (correct)

Which component is crucial for the controlled expression of a target gene in an expression vector?

Transcriptional and translational control sequences (correct)

Ori site

Lac Z′ gene

Bacterial chromosome

What role does the T7 RNA polymerase gene play in expression vectors?

Provides antibiotic resistance

Facilitates DNA replication

Controls transcriptional activity (correct)

Enhances stability of plasmid

In the context of pBACs, what is the significance of them being accommodated as single copies in each bacterium?

Facilitates chromosome mapping Signup and view all the answers

What percentage of total cellular protein can the target protein produced by expression vectors reach?

40% Signup and view all the answers

What is the purpose of including a multiple cloning site (MCS) in a plasmid vector?

To provide several unique restriction sites for DNA insertion Signup and view all the answers

Which of the following statements is NOT true regarding the insertion of foreign DNA into plasmid vectors?

A single restriction digest is less error-prone than a double restriction digest. Signup and view all the answers

What is the primary function of antibiotic resistance in molecular cloning?

To eliminate untransformed host cells from the culture Signup and view all the answers

How does blue-white screening differentiate between cloned bacteria?

By producing differently colored colonies based on lac Z' gene activity Signup and view all the answers

What is a disadvantage associated with using the single restriction method for inserting DNA?

It can lead to the insertion of DNA in both orientations. Signup and view all the answers

Why is it important for restriction sites in a plasmid to not be located in essential replication regions?

To ensure that plasmid replication can occur regardless of insertions Signup and view all the answers

Which combination of restriction enzymes is suggested for double restriction cloning?

BamHI and EcoRI Signup and view all the answers

What type of DNA is synthesized from an mRNA template by reverse transcriptase?

Single-stranded DNA Signup and view all the answers

Which property is NOT essential for an ideal bacterial plasmid vector?

Exoribonuclease activity Signup and view all the answers

Which of the following plasmids is known for having a high copy number?

pUC18 Signup and view all the answers

What is the purpose of the 3′-5′ exoribonuclease activity of reverse transcriptase?

To degrade RNA in DNA-RNA hybrids Signup and view all the answers

What is the initial product synthesized from mRNA by reverse transcriptase?

cDNA Signup and view all the answers

In plasmids, what does 'stringent control' refer to?

Replication linked to host chromosome Signup and view all the answers

Which of the following methods is NOT a source of foreign DNA for cloning?

Plasmid DNA Signup and view all the answers

Which enzyme is primarily responsible for the synthesis of the second strand during the cDNA synthesis?

Klenow fragment of DNA polymerase I Signup and view all the answers

What characteristic of early plasmid vectors like pBR322 limits their replication?

Low copy number Signup and view all the answers

What is the result of mixing a 15 kbp fragment of foreign DNA with λ DNA cut by the same restriction enzyme?

It results in a chimeric DNA ready for packaging into phage heads. Signup and view all the answers

Which of the following describes the role of the lac Z′ gene in the selection process?

It helps distinguish colonies with the DNA insert as white colonies. Signup and view all the answers

What feature distinguishes cosmids from regular plasmids?

They contain a cos site for packaging into phage heads. Signup and view all the answers

What is the minimum distance between cos ends necessary for cosmids to be packaged into phage heads?

38 kbp Signup and view all the answers

What is the primary advantage of using BACs in cloning?

They allow for the incorporation of large DNA pieces. Signup and view all the answers

What is the purpose of the cohesive ends in linear λ DNA?

To enable annealing and ligation when entering E. coli. Signup and view all the answers

How do transformants containing lambda with an insert appear when cultured on agar plates?

They appear as white plaques among blue colonies. Signup and view all the answers

Which statement best characterizes the function of the F plasmid?

It ensures even distribution of plasmids during bacterial division. Signup and view all the answers

What type of molecules do cosmids combine advantages from?

Plasmids and phage vectors. Signup and view all the answers

What color do colonies turn if they contain plasmids without DNA inserts when grown on X-gal plates?

Blue Signup and view all the answers

What is the role of isopropyl β-D-1-thiogalactopyranoside (IPTG) in the context of the Lac operon?

It serves as an inducer. Signup and view all the answers

Which bacteriophage was one of the first to be used in cloning?

Bacteriophage lambda (λ) Signup and view all the answers

What is the maximum length of DNA that can typically be cloned using bacteriophage lambda?

18 kbp Signup and view all the answers

What happens when lambda bacteriophage grows lysogenically?

It inserts its DNA into the host genome. Signup and view all the answers

What is the linear genome size of bacteriophage lambda?

48.5 kbp Signup and view all the answers

Which of the following is NOT a function of the non-essential genes in lambda's genome?

Retaining the lytic growth capability Signup and view all the answers

What indicates the presence of foreign DNA in transformant colonies on X-gal plates?

White colonies Signup and view all the answers

What is a significant advantage of using bacteriophage vectors over plasmids?

Bacteriophages can introduce larger DNA fragments. Signup and view all the answers

What happens if DNA being packaged into the lambda head is less than 38 kbp?

It will not package into the head. Signup and view all the answers

Study Notes

RNA-dependent DNA Polymerase (Reverse Transcriptase)

Catalyzes the synthesis of single-stranded DNA from mRNA templates
Needs a primer, like regular DNA polymerases
Possesses 5'-3' exoribonuclease activity and 3'-5' exoribonuclease activity. This specifically degrades RNA in DNA-RNA hybrid molecules.
Used to transcribe mRNA into double-stranded DNA (dsDNA)
This dsDNA can be inserted into prokaryotic vectors.
First-strand cDNA is made; then, RNA is degraded by alkali or ribonuclease H
Second-strand synthesis uses Klenow fragment of DNA polymerase I or reverse transcriptase itself. The cDNA acts as both primer and template, forming a hairpin structure.
Reverse transcriptase lacks proofreading ability.

Cloning and Expression Vectors

Plasmids: Named using uppercase letters and numbers, e.g., pBR322. Lowercase "p" indicates plasmid.
Early plasmid vectors often have low copy numbers (one or two copies per cell). Higher copy number plasmids, like pUC18 and pUC19, have >500 copies per cell. Replication is either "relaxed" (uncoupled from host chromosome) or "stringent" (coupled).
Ideal plasmids need:
- Replication origin (ori) for independent replication of the plasmid and inserted DNA
- Selection marker(s) to detect recombinants
- Multiple cloning site (polylinker) with unique restriction enzyme sites
- Easy extraction from the host cell

Insertion of Foreign DNA into a Plasmid Vector

Single Restriction: Plasmid and foreign DNA have compatible sticky ends, allowing ligation. Can insert in either orientation. Multiple fragments possibly inserting into the excised plasmid.
Double Restriction: Uses two different restriction enzymes (e.g., BamHI and EcoRI) for both plasmid and foreign DNA. Cleaving both creates specific overhangs/sticky ends. Improves control over the orientation of the insert relative to the host gene.

Bacterial Transformation

Introducing recombinant plasmid into bacterial cells using either calcium phosphate treatment or electroporation to transiently make the bacterial membrane porous.
Three types of bacterial cells after transformation:
- No plasmid
- Plasmid without foreign insert
- Plasmid with foreign insert
Selection based on antibiotic resistance or blue-white screening (using X-gal).

Bacteriophage Vectors

Efficient for introducing foreign DNA into bacteria (compared to transformation)
Lambda (λ) phage:
- Can grow lysogenically (insert DNA into host chromosome) or lytically (make copies and package within phage particles).
- Genome size allows cloning of ~18kb inserts.
- Important to consider the necessary restriction sites and the ability to maintain the replication of the fragment.
- Selectable marker for transformants (e.g., white vs. blue colonies based on disruption of lacZ encoding gene).

Cosmids

Plasmid with a cos site (cohesive ends for packaging).
Can accommodate larger inserts (33–47 kb) packaged efficiently into phage heads.

Bacterial Artificial Chromosomes (BACs)

Based on the fertility plasmid (F plasmid)
Contain partition genes for even plasmid distribution
Can accommodate large DNA inserts (> 50 kb)

Expression Vectors

Designed for high-level expression of a target gene's protein product.
Typically plasmid-based.
Often use a highly efficient and tightly controlled T7 RNA polymerase gene expression system.
Cloning structural genes into a plasmid that has the appropriate transcriptional and translational controls to express the protein. The plasmid is then introduced into a host cell that already has the appropriate polymerase encoded.

Studying That Suits You

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

Description

This quiz covers key concepts related to RNA-dependent DNA polymerase, also known as reverse transcriptase, and its role in synthesizing cDNA from mRNA templates. Additionally, the quiz explores the use of plasmids as cloning and expression vectors, highlighting their characteristics and applications in molecular biology. Test your knowledge on these fundamental topics in biotechnology.