Chapter 18 – Recombinant DNA and Biotechnology

What is recombinant DNA?

A DNA molecule made in the lab using DNA from two or more different sources. Sources can be from different chromosomes, different individuals of the same species, or completely different organisms.

What are restriction enzymes and what types of ends do they create?

Restriction enzymes cut DNA at specific palindromic sequences. They produce:

• Blunt ends: straight cuts

• Sticky ends: staggered cuts with single-stranded overhangs that can hydrogen bond with complementary sequences

What is the role of DNA ligase in recombinant DNA?

DNA ligase seals nicks in the sugar-phosphate backbone, forming covalent bonds and creating a stable, continuous DNA molecule.

Why do scientists insert DNA into cells?

To clone genes, produce proteins, or create organisms with new phenotypes by making millions of identical copies of a DNA sequence.

What is the difference between transformation and transfection?

• Transformation: inserting DNA into prokaryotic cells

• Transfection: inserting DNA into eukaryotic (animal) cells

What is a selectable marker and why is it used?

A gene (often antibiotic resistance) used to identify transgenic cells. Only cells that have successfully taken up foreign DNA survive under selection.

Name two main ways to make transgenic multicellular organisms.

• Zygotic injection: inject transgene into fertilized egg → all cells transgenic

• Transformed cell injection: transform totipotent stem cells, inject into embryo → mosaic organism

Why are plasmids useful as vectors?

They are small, replicate independently, have unique restriction sites, selectable markers, and an origin of replication (ori).

Give examples of reporter systems used to identify recombinant cells.

• Antibiotic resistance

• lacZ (β-galactosidase → blue/white screening)

• GFP (fluorescent under UV light)

What is a genomic library?

A collection of all DNA fragments of an organism’s genome, each inserted into a vector and transformed into host cells.

What is a cDNA library?

A collection of DNA copies of mRNA (only protein-coding sequences), made using reverse transcription, representing genes actively transcribed in a tissue.

How does PCR work and when is it useful?

Polymerase Chain Reaction amplifies specific DNA sequences using primers, DNA polymerase, and nucleotides. Useful when the sequence is known and DNA libraries are unnecessary.

What is the role of synthetic DNA in biotechnology?

Chemically synthesized DNA is used for making mutated genes, artificial genes, primers, or entire synthetic genomes.

How do scientists choose which genes to study?

By genetic mapping (linking genes to disorders) or analyzing gene expression patterns (mRNA levels in tissues, developmental stages, or disease states).

What is a DNA microarray and how does it work?

A glass slide with thousands of DNA sequences that hybridize with RNA from a sample. Color coding indicates expression levels: red = high in one tissue, green = high in another, yellow = similar in both.

Name three methods to modify gene expression after transcription.

• Reporter genes → track expression

• RNA interference (RNAi) → block mRNA translation (miRNA/siRNA)

• Antisense RNA → synthetic RNA binds target mRNA to block translation

Compare RNAi and CRISPR.

RNAi works post-transcriptionally by degrading or blocking mRNA, leading to gene knockdown, while CRISPR acts at the DNA level to cut, mutate, or replace genes, resulting in knockout, knockdown, or precise gene edits.

What are expression vectors and why are they used?

Plasmids with sequences to promote gene expression (promoter, poly-A tail, signal sequences) used to produce proteins in host cells.

What is synthetic biology?

Creating artificial genomes and inserting them into cells to produce organisms with new functions (biofuels, plastics, drugs), requiring careful design of genes and promoters.