9. Recombinant DNA Technology

What is Recombinant DNA Technology?

A technique that allows scientists to isolate a gene from any organism and join genes from different sources to create recombinant DNA.

What are the purposes of Recombinant DNA Technology?

Create infinite amounts of target DNA, express genes as proteins, produce probes, deduce function.

What are the basic tools of Recombinant DNA Technology?

Restriction enzymes, DNA ligase, and vectors such as plasmids.

What are restriction enzymes?

Enzymes that cleave DNA at specific sites.

What is a characteristic of restriction enzyme recognition sites?

Restriction enzymes recognize palindromic sequences of four to eight base pairs.

What are frequent cutters in the context of restriction enzymes?

Restriction enzymes that cut at four base pair sites will cut more often than those that cut at eight base pair sites.

What are the possible products of a restriction enzyme cut?

5' sticky ends, 3' sticky ends, and blunt ends.

What is the importance of sticky ends?

Sticky ends produced by restriction enzymes can anneal to each other due to complementary overhangs.

What role does DNA ligase play in recombinant DNA technology?

DNA ligase joins the two annealed pieces of DNA together by creating a phosphodiester bond.

How many restriction enzymes have been discovered?

3000 restriction enzymes that recognize 230 different DNA sequences.

What are vectors in the context of recombinant DNA?

DNA sequences used to replicate genes of interest.

What property is most important for vectors?

Self-replication.

Why are smaller vectors preferred in genetic engineering?

Smaller vectors are easier to manipulate and less fragile than larger vectors.

What types of vectors are commonly used in genetic engineering?

Plasmids, bacteriophage, cosmids, bacterial artificial chromosomes, and yeast artificial chromosomes.

What features do plasmids have?

Origin of replication, antibiotic resistance gene, various restriction enzyme cut sites, and positive selection genes.

How do plasmids facilitate antibiotic resistance?

They carry antibiotic resistance genes, allowing the selection of bacteria that contain the plasmid by killing others that do not.

What is transformation in the context of plasmid insertion?

The insertion of a foreign plasmid into a cell.

What is one method used to introduce plasmids into cells?

Electroporation, which opens temporary pores in cell membranes.

What are the three possible outcomes after inserting a plasmid into a bacteria?

Bacteria with no plasmid, bacteria with plasmid but no insert, bacteria with plasmid that has the insert.

How can transformed bacteria be selected?

Using media with ampicillin and substrates like x-gal and IPTG to differentiate between active and inactive β-galactosidase.

What is one application of Recombinant DNA Technology?

Making microbes produce various protein products like foods, vaccines, and antibiotics.

What is an example of a pharmaceutical product derived from rDNA?

Human insulin, produced by E. coli.

What was one significant advantage of recombinant technology over past methods of producing human growth hormone?

It provides a pure and cost-effective product without the risk of transmitting diseases.

What does CRISPR stand for?

Clustered Regularly Interspaced Short Palindromic Repeats.

What is the function of the Cas9 protein in CRISPR?

It is a nuclease that cleaves DNA at specific target locations.

What is the role of crRNA in CRISPR technology?

It guides Cas9 to the target sequence via base pair matching.

What is homology directed repair (HDR)?

A method where template DNA is added for precise gene reattachment by ligase.

What is non-homologous end joining (NHEJ)?

A process that allows for the repair of double-stranded breaks in DNA.

What are the advantages of using CRISPR technology?

Ease of use, high transformation efficiency, and flexibility.

What is a limitation of CRISPR technology?

It relies on DNA repair machinery and can potentially cause unwanted mutations.

What is a repeat expansion mutation?

A mutation that increases the number of times a region of DNA is repeated.

What is a missense mutation?

A single base pair change that alters the amino acid sequence.

What is a nonsense mutation?

A base pair change that creates a premature stop codon in the protein sequence.

What does insertion mutation involve?

The addition of one or more nucleotides into the DNA sequence.

What does deletion mutation involve?

The removal of one or more nucleotides from the DNA sequence.

What causes frameshift mutations?

Insertions or deletions that change the reading frame of the gene.

What is a duplication mutation?

A mutation that results in the duplication of a segment of DNA.

What can be said about the majority of restriction enzymes?

Most are dimers consisting of two identical subunits.

What is the significance of choosing the right restriction enzyme?

It determines how the gene of interest is recombined with a vector.

Which technology has been noted for producing a wide range of proteins?

Recombinant DNA Technology.

What aspect of plasmids helps avoid degradation?

Their circular structure.

How can we ensure specific insertion orientation in plasmid construction?

By using two different restriction enzymes to create distinct sticky ends.