Core Concepts of Genetics, DNA Technology, and Profiling Methods

Principles of Inheritance and Genetics

Basic Genetic Terminology

Diploid Organisms: Organisms possessing two sets of chromosomes and two alleles (forms) of each gene.

• Homozygous: Having two identical alleles for a trait (e.g., C^RC^R).
• Heterozygous: Having two different alleles for a trait (e.g., C^RC^r).

Mutation: A change in the amount or the chemical structure of DNA.

• Chromosomal Mutation: A difference in the number or structure of chromosomes.
• Gene Mutation: Changes within a gene (e.g., substitution, deletion, addition).

Phenotype: The observable characteristics (e.g., tall).

Genotype: The genetic makeup (e.g., Tt).

Gamete: A haploid reproductive cell (e.g., T & t).

Patterns of Inheritance

Codominance: Both alleles are expressed in the heterozygote.

• C^RC^R: Red
• C^RC^r: Pink
• C^rC^r: White

Dihybrid Cross: Involving two traits (e.g., TtPp) resulting in four possible gametes: TP, Tp, tP, tp.

Sex Linkage: Inheritance patterns determined by genes located on the sex chromosomes.

• Male: XY
• Female: XX

Genetic Engineering and Recombinant DNA

Recombinant DNA Technology Steps

1. Locate a specific gene in the donor cell.
2. Isolate this gene in a piece of donor DNA.
3. Modify the donor DNA (e.g., inserting it into a vector).
4. Transfer the modified donor DNA into host cells.

Methods for Gene Isolation (Steps 1 & 2)

Genetic Probes

Genetic probes consist of a single strand of DNA that contains a known sequence of bases. It is labeled with a radioactive marker. The bases in the genetic probe combine with the complementary bases on the donor DNA, revealing the position of the gene. Once located, the gene can be extracted.

Reverse Transcription

In the presence of a high amount of mRNA, if it can be isolated, its complementary DNA (cDNA) can be synthesized from this process. This is called reverse transcription because mRNA acts as a template for DNA synthesis.

The process occurs after the mRNA strand has been copied into cDNA. The RNA is removed, and a second strand of DNA is made by adding the enzyme DNA polymerase and more nucleotides. The result is an identical double-stranded DNA molecule.

Artificial DNA Synthesis

If the base sequence is known, it can be deduced from the amino acid sequence of the protein.

Key Enzymes and Vectors

Restriction Endonucleases and DNA Ligase

• Restriction Endonucleases: Enzymes that cut DNA at specific recognition points.
• DNA Ligase: An enzyme that joins or “sticks” the cut DNA fragments together.

Plasmids (Vectors)

Plasmids are small, circular rings of DNA found in some bacteria. They replicate independently and can be transferred from one bacterial cell to another or to plant cells. Plasmids are used as vectors: a part of this DNA is cut (using restriction endonucleases) and the desired DNA fragment is inserted (using DNA ligase) so as to transfer the DNA.

DNA Profiling Techniques

DNA Used in Profiling

DNA used in profiling contains hypervariable regions, which have repeating nucleotide sequences called core nucleotide sequences.

Steps in DNA Profiling

1. Extraction: DNA is dissolved by mixing a tissue sample with a solvent.
2. Digestion: Restriction endonucleases cut the DNA, producing fragments, some of which contain the hypervariable regions.
3. Separation (Electrophoresis):
   1. Fragments are placed in a gel, and an electric current is applied.
   2. DNA fragments move because they are negatively charged. Smaller fragments move faster.
   3. The separated fragments are then printed onto a nylon membrane (blotting).
4. Hybridization: Single-stranded DNA on the nylon membrane is mixed with probes that have a fluorescent or visible marker, allowing the specific hypervariable regions to be visualized.