genomes

Genome

The genome is the complete set of genetic material in an organism.

Human Genome Size

The human genome has ~3.3 billion base pairs.

Mitochondrial DNA (mtDNA)

Found in mitochondria, it is a circular DNA molecule with 16,569 base pairs.

Chloroplast DNA (cpDNA)

In plants, some also have chloroplast DNA.

Importance of Genome Sequencing

Knowing the genome sequence allows comparison of genes and regulatory sequences between individuals, understanding of the genetic basis of disease, and determination of evolutionary relationships between species.

Coding Regions

Coding regions are parts of the genome that are transcribed and translated.

Exons

Exons are coding sequences that remain in mRNA.

Introns

Introns are non-coding sequences removed during RNA splicing.

Gene Expression

Not all genes are active all the time; around 20,000-25,000 genes in the human genome.

Housekeeping Genes

type of genes that are always on and are specifically regulated.

Pleiotropic Genes

Pleiotropic genes are expressed in multiple tissues and have multiple, distinct effects.

Non-Coding Regions

Non-coding regions include introns and intergenic DNA (regions between genes). once called junk DNA but are now known to have regulatory functions.

MicroRNAs (miRNAs)

Some introns encode microRNAs, which form part of RISC (RNA-Induced Silencing Complex) and regulate translation.

Gene Technologies

Gene technologies modify genomes using artificial DNA manipulation.

Recombinant DNA (rDNA)

Recombinant DNA is created by combining DNA from two or more sources.

Steps of Recombinant DNA Technology

Recombinant DNA and gene cloning involve five main steps: Isolation, Cleavage, Ligation, Transformation, and Selection/Screening.

Polymerase Chain Reaction (PCR)

PCR is an in vitro technique to amplify DNA sequences exponentially.

Taq Polymerase

Taq polymerase is isolated from Thermus aquaticus and is thermostable, allowing automation.

Reverse Transcription

Reverse transcription converts mRNA to complementary DNA (cDNA) using reverse transcriptase.

cDNA

cDNA reflects expressed genes in a specific tissue/time.

Restriction Enzymes

Endonucleases that cut DNA at specific sequences (restriction sites, usually 4-8 bp).

Sticky ends

Staggered cuts → overhangs that pair easily.

Blunt ends

Straight cuts → no overhangs.

step 3-Ligation

DNA fragments joined by DNA ligase (forms covalent bonds between nucleotides).

Transformation

Introduction of recombinant DNA into host cells (e.g., bacteria).

Heat Shock

Cells incubated in Ca²⁺ at 0°C → briefly heated (~40°C).

Electroporation

Electric pulse temporarily opens pores in the cell membrane.

Selection and Screening

Identifies cells successfully transformed with recombinant DNA.

Vector

A vector carries foreign DNA from one organism (donor) into another (host).

Plasmids

Naturally occurring, circular dsDNA in bacteria.

Cloning vectors

Isolate and amplify DNA (e.g., pUC, pBR322).

Expression vectors

Express genes as proteins (e.g., pET, pcDNA3.1).

Origin of replication (ori)

Ensures replication in host.

Selectable marker

E.g., Ampicillin resistance.

Promoter region

Bacterial or mammalian, depending on host.

Applications of Recombinant DNA Technology

Major Uses: Study of gene structure and expression, production of pharmaceuticals, creation of GMOs, gene therapy and disease research.

Insulin

Hormone that regulates blood glucose.

Insulin Structure

Two chains: A (21 amino acids) and B (30 amino acids).

E. coli

Simple, well-understood genetics; fast growth, low cost, easy to culture.

Mitochondrial DNA

Circular, maternally inherited DNA with 37 genes.

Recombinant DNA

Combines DNA from different sources using enzymes.

PCR

Amplifies DNA in vitro using heat-stable polymerase.

Applications

Insulin, GMOs, vaccines, gene therapy.

what do genes consist of

introns and exons

Mitochondrial DNA (mtDNA)

DNA found in the mitochondria. Mitochondrial DNA is inherited only from the mother.