Recombinant DNA and Protein Expression

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Questions and Answers

What is the primary feature of Type II restriction enzymes?

They can only target sequences longer than 8 bases.

They recognize random DNA sequences.

They always cut DNA at single-stranded regions.

They recognize palindromic sequences. (correct)

Which of the following exemplifies the nomenclature for restriction enzymes?

E. coli (correct)

M. influenzae

B. cereus

R. salmonella

What prefixes are used to distinguish between restriction and modification enzymes?

E for enzyme and R for restriction.

M for modification and D for DNA.

R for restriction and M for modification. (correct)

C for cleavage and M for methylation.

Which of the following statements is false regarding Type II restriction enzymes?

They require ATP for their activity. Signup and view all the answers

What characteristic do the 5' termini generated by certain restriction enzymes have?

They are capable of associating due to hydrogen bonding. Signup and view all the answers

What is a primary application of genetically engineered organisms?

Enhancing natural pest resistance in crops Signup and view all the answers

Which protein is specifically associated with the treatment of heart attack therapy?

Streptokinase Signup and view all the answers

What is included in the regulations monitoring recombinant DNA (rDNA)?

International and national rules Signup and view all the answers

What is one function of recombinant antibodies?

Diagnosing infectious diseases Signup and view all the answers

Which of the following is NOT a recombinant DNA product?

Natural animal hormones Signup and view all the answers

What is one benefit of using the gene for pest resistance in plants?

Minimized environmental impact from conventional farming Signup and view all the answers

What step follows the extraction of DNA from blood or tissue in basic research?

PCR and restriction digestion Signup and view all the answers

Which recombinant product is used for treating growth-related issues?

Human growth factor Signup and view all the answers

What was the key significance of Werner Arber's discovery in 1962?

Proof of the existence of molecular scissors that can cut DNA Signup and view all the answers

What is the main function of restriction endonucleases (REs)?

To cut DNA at specific sequences Signup and view all the answers

Type I restriction enzymes have which of the following characteristics?

They require methylation at the host specificity site. Signup and view all the answers

What distinguishes Type II restriction enzymes from Type I and III?

Type II enzymes are single subunit enzymes. Signup and view all the answers

How do Type III restriction enzymes differ in their cutting mechanism?

They cut DNA 24-26 bp from recognition sequences. Signup and view all the answers

What is a requirement for Type I restriction enzymes to perform their function?

Presence of Mg2+ and S-adenosyl methionine Signup and view all the answers

Which statement best describes the specificity of Type II restriction enzymes?

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

Why are Type II restriction enzymes commonly used in gene cloning?

They have high specificity for their recognition sequences. Signup and view all the answers

What is the purpose of using cDNA in genetic research?

To synthesize a complementary copy of mRNA Signup and view all the answers

Which of the following steps is NOT involved in the isolation of genomic DNA?

Cloning of cDNA in host cells Signup and view all the answers

What do exons refer to in the context of RNA processing?

The coding regions that will be expressed in the final mRNA Signup and view all the answers

What is the primary reason for using isopropanol during genomic DNA isolation?

To precipitate genomic DNA from a solution Signup and view all the answers

Which of the following processes does not occur during cDNA synthesis?

Transcription of DNA to mRNA Signup and view all the answers

What is the role of Proteinase K in genomic DNA isolation?

To digest proteins that may contaminate the DNA Signup and view all the answers

What occurs after the cellular lysis step in genomic DNA isolation?

Protein precipitation using chloroform Signup and view all the answers

What is the significance of performing an ethanol wash after precipitating genomic DNA?

To remove impurities and salts from the DNA Signup and view all the answers

What is the primary function of DNA ligases?

To join DNA molecules that are part of a double helix Signup and view all the answers

What significant achievement is Arthur Kornberg known for?

Using ligase to create biologically active viral DNA Signup and view all the answers

Which type of DNA ligase is commonly used in laboratory settings?

ATP dependent DNA ligase from T4 phage Signup and view all the answers

In the context of DNA polymerases, which statement is correct?

DNA polymerase can add nucleotides to a pre-existing 3'-OH group. Signup and view all the answers

How do RE (Restriction Enzymes) function in conjunction with ligases in cloning?

RE cut DNA into smaller pieces, and ligases join those pieces together. Signup and view all the answers

What is the direction of elongation for new DNA strands added by DNA polymerase?

5' to 3' Signup and view all the answers

What is required for DNA polymerase to initiate nucleotide addition?

A pre-existing 3'-OH group Signup and view all the answers

Why is DNA ligase critical for constructing artificial viral DNA?

It allows the joining of fragmented DNA into continuous loops. Signup and view all the answers

Study Notes

Gene of Interest and Protein Expression

Genes of interest can be linked to specific diseases, with associated defects.
Copies of these genes are used in basic research and various applications, leading to protein harvesting.
Organisms can be engineered for purposes like insulin production and insect resistance, referred to as "pharm" animals.

Recombinant DNA Products

Notable recombinant DNA products include:
- Human Insulin
- Human growth factor
- Hepatitis B virus vaccine
- Streptokinase, which dissolves blood clots
- Recombinant antibodies and enzymes

Regulations for Recombinant DNA

International and national regulations oversee organisms that use recombinant DNA (rDNA).
Special permissions and facilities are required for the use of rDNA organisms and products.

DNA Cloning Process

DNA cloning involves several steps:
- Extraction of DNA from blood or tissue.
- PCR/RE digestion of DNA and vector.
- Ligation of DNA into the vector.
- Transformation into a suitable host.
- Screening for positive clones.
- Expression of desired products in the host.

Nucleic Acids Isolation

Genomic DNA is the complete DNA content used for full coding regions of prokaryotes.
cDNA is synthesized from mRNA, essential for eukaryotic coding regions.

Genomic DNA Isolation

Involves lysis of cells with detergent, protein digestion, and DNA precipitation.
Key steps:
- Use of Proteinase K to digest proteins.
- DNA is purified using phenol, chloroform, or salt, followed by isopropanol precipitation.

cDNA Synthesis

Begins with purification of mRNA followed by synthesizing first and second strands of cDNA.
Cloning of cDNA is the final step for applications.

Transcription and RNA Processing

Exons represent coding regions separated by non-coding introns.
Processed mRNA results from intron splicing, leading to the final transcript.

Molecular Scissors: Restriction Endonucleases

Discovery in 1962 by Werner Arber, who identified enzymes that cut DNA.
Restriction endonucleases (REs) recognize specific sequences to cleave DNA.
REs are critical for recombinant DNA technology, categorized into:
- Type I: Multifunctional, random cutting.
- Type II: Specific, used in cloning.
- Type III: Recognizes non-palindromic sequences, cut near recognition sites.

Characteristics of Type II Restriction Enzymes

Single subunit enzymes requiring Mg2+ and recognizing palindromic sequences (4-8 nucleotides).
Specific cleavage closely at recognition sites, facilitating gene cloning.

Nomenclature of Restriction Enzymes

Named after the genus and species of the host organism, with modifiers for strains.
Prefix 'R' for REs and 'M' for modification enzymes.

DNA Ligases in Cloning

Used to join DNA fragments, with T4 phage ligase being commonly utilized.
Ligases enable the formation of continuous DNA from cut fragments, essential for creating recombinant DNA.

DNA Polymerase Function

DNA polymerase adds nucleotides in a 5’-3’ direction and requires a primer for initiation.
Proofreading mechanisms are in place for error correction during DNA strand synthesis.

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Description

This quiz covers the essentials of recombinant DNA technology, including gene manipulation, protein expression, and the applications of genetically engineered organisms. Additionally, it explores notable recombinant products and the regulations surrounding their use. Test your knowledge on DNA cloning processes and the significance of these biotechnological advancements.