6.3

Gene cloning was made possible by the discovery of restriction enzymes that cut DNA molecules at specific locations.
Bacteria utilize restriction enzymes to cut foreign DNA, including from phages or other bacteria.
Restriction Enzymes: Recognize short DNA nucleotide sequences and cut at specific points in these sequences.
Bacteria protect their own DNA through methylation.

Each restriction enzyme cleaves a specific sequence of bases, referred to as the restriction site.
- These sequences are typically symmetrical, ranging from four to eight bases in length, on both strands running in opposite directions.
- Example:
- If the restriction site on one strand is 3’-CTTAAG-5’, then the complementary strand would be 5’-GAATTC-3’.
Inverted Repeats: Identical copies of a DNA molecule will always yield the same set of restriction fragments when exposed to a particular enzyme.

Restriction Enzymes and DNA Ligase can be used to create recombinant DNA, which is DNA spliced together from two different sources.
Restriction enzymes cut the bonds of both strands, typically in a staggered manner, creating sticky ends on each cut piece (referred to as restriction fragments).
Sticky ends form hydrogen bonds with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme.
This requirement for the same restriction enzyme assures compatibility between the DNA fragments being joined.

Several methods can be employed to get a gene into a cell. If an object (e.g., a virus) acts as a delivery mechanism, that object is termed a vector.
Plasmids are often used as vectors to introduce a gene into a bacterial cell.

The process of cloning a human gene in a bacterial plasmid can be encapsulated in five major steps:
Identifying Cell Clones: Using nucleic acid probes to find the bacterial cell that took up the desired gene, where a radioactive isotope or fluorescent protein labels the probe.
- Laboratory Process:
1. Transfer cells to a filter.
2. Treat cells on the filter to denature the DNA.
3. Add the probe to the filter for hybridization.
4. Conduct autoradiography to visualize the colonies containing the gene of interest.

DNA cloning is deemed the best method for preparing large quantities of a specific gene or DNA sequence.
When the source of DNA is limited or impure, PCR offers a quicker and more selective alternative, albeit for small sequences.
PCR is capable of amplifying any small DNA segment without requiring cells.

Cycle Process: The PCR process entails a three-step cycle (heating, cooling, and replication) that is repeated multiple times to achieve exponential growth of DNA molecules.
Taq Polymerase: Key to PCR automation; isolated from high-temperature-resilient bacteria, it withstands the heat required to denature DNA strands.
Probes vs. Primers: Both are short nucleotide strands with bases complementary to those of another strand, but primers initiate replication while probes facilitate the localization of specific DNA segments.

PCR was developed in 1985 by Kerry Mullis and has had immense impacts on biological research and technology.
PCR applications include:
- Amplifying ancient DNA from a 40,000-year-old wooly mammoth.
- Isolating DNA from small samples found at crime scenes, such as blood or semen.
- Rapid prenatal diagnosis from a single embryonic cell.
- Amplifying viral genes, e.g., from cells infected with HIV.

After cutting DNA with restriction enzymes, fragments can be separated by size using gel electrophoresis, producing a specific band pattern.
This method allows for the recovery of pure samples of individual fragments.
The mobility of fragments in the gel is influenced by size, with shorter fragments moving further than larger ones.

DNA Profiling: Identifies criminals or determines paternity.
- Samples from different individuals are treated with the same restriction enzyme, and fragments are separated via gel electrophoresis.
- Results indicate differing restriction patterns, aiding in identification.

Southern Blotting examines differences in noncoding DNA.
Restriction Fragment Length Polymorphisms (RFLPs) can indicate genetic locations (loci) and serve as genetic markers for identification purposes.

An ambitious project initiated in 1990 aimed at mapping the entire human genome and determining nucleotide sequences for each chromosome.

Gene therapy harnesses genetic manipulation techniques to treat diseases by inserting normal alleles into afflicted somatic cells.
- Permanent alteration requires targeting cells that will multiply throughout the patient's life.
- RNAi is one type of gene therapy.
Applications include treating cystic fibrosis with engineered retroviruses to deliver healthy genes to respiratory cells, alongside other diseases such as Parkinson's.

Genetic engineering extends to environmental applications:
- Genetically engineered microbes could extract heavy metals for safe recovery and cleanup of toxic waste.
- Oil-eating microbes may assist in cleaning oil spills.

Reproductive Cloning: Created through somatic cell nuclear transfer (SCNT); Dolly the sheep was the first clone.
- Clones are genetically identical to the original organism, resulting in exact copies.
- Cloning plants and some animals, such as starfish, is simpler compared to others.
Therapeutic Cloning: Focuses on creating embryos for stem cell harvesting, with significant ethical and moral considerations.

Stem cells can be sourced from:
- Embryos (via therapeutic cloning)
- Umbilical cord blood or placenta of newborns
- Certain adult tissues (e.g., bone marrow)
Various methods, such as SCNT, involve significant ethical debates due to insemination of embryos.

Advancements include CRISPR technology for delivering genes and RNA interference for degrading unwanted RNA sequences.
- CRISPR, which has components that create immunity in prokaryotes, has gained widespread attention for its potential in genetic modifications.