Crizelda D. Liwanag, MSc, RMT

Genetic Manipulations

Applications in Diagnosis

Applications in Diagnosis

1. Detection of DNA1. Electrophoresis

2. Southern Blotting

2. PCR

3. DNA Fingerprinting

4. DNA Sequencing

1. Chromosomes, which range in size from 50 million to 250 million bases, must first be broken into much shorter pieces (subcloning step).

2. Each short piece is used as a template to generate a set of fragments that differ in length from each other by a single base that will be identified in a later step (template preparation and sequencing reaction steps).

3. The fragments in a set are separated by gel electrophoresis (separation step).

4. New fluorescent dyes allow separation of all four fragments in a single lane on the gel.

5. The final base at the end of each fragment is identified (base-calling step). This process recreates the original sequence of As, Ts, Cs, and Gs for each short piece generated in the first step.

6. Automated sequencers analyze the resulting electropherograms, and the output is a four-color chromatogram showing peaks that represent each of the four DNA bases.

7. After the bases are "read," computers are used to assemble the short sequences (in blocks of about 500 bases each, called the read length) into long continuous stretches that are analyzed for errors, gene-coding regions, and other characteristics.

Genetic Manipulations

Applications in Therapy

Applications in Therapy

1. Mutagenecity and anti-mutagenecity assays

2. Recombinant DNA technology

3. Gene transfer and movement

4. Gene therapy

5. RNA interference

6. Stem cell and organ cloning

7. GMO’s

Gene therapy

Gene therapy

• Insert a working gene

Gene therapy

• Use virus

Gene therapy

• Like an envelope carrying a letter

Gene therapy

• Gene randomly inserts itself into genome

Gene therapy

• It can now be read (correct instruction)

RNA interference

RNA interference

• Suppressors

RNA interference

• From viruses (replace suppressors)

RNA interference

• New genes cause cancer

RNA interference

• Small interfering RNA binds to mRNA of infected genes

Stem cell therapy

• Stem cells

Stem cell therapy

• Trick stem cells of a different organ into regenerating an organ

Stem cell therapy

• Damaged areas of the heart

Stem cell therapy

• Black dots representing areas injected with stem cells

Stem cell therapy

• Stem cells from femur BM are transferred to the heart and they mimic the cells surrounding them

Stem cell therapy

• Purple area shows healthy heart

Stem cell therapy

• Frog’s eyes grown from stem cells

Stem cell therapy

• 1st to grow sensory organs from embryonic stem cells

Stem cell therapy

• Frogs that cannot see are lighter in color

Stem cell therapy

• Clone of own embryo (to solve histocompatibility and immunologic tolerance problems)

Stem cell therapy

Embryonic stem cell cloning

• Co-creator of DOLLY

Embryonic stem cell cloning

• Trying to clone a human being

Believes cloning will be done

What we still DON’T know, even with the full human sequence in hand:

• Gene number, exact locations, and functions

• Gene regulation • DNA sequence organization • Chromosomal structure and organization • Noncoding DNA types, amount,

distribution, information content, and functions

• Coordination of gene expression, protein synthesis, and post-translational events

• Interaction of proteins in complex molecular machines

What we still DON’T know, even with the full human sequence in hand:

• Predicted vs experimentally determined gene function

• Evolutionary conservation among organisms • Protein conservation (structure and function) • Proteomes (total protein content and function) in

organisms • Correlation of SNPs (single-base DNA variations

among individuals) with health and disease • Disease-susceptibility prediction based on gene

sequence variation • Genes involved in complex traits and multigene

diseases • Complex systems biology including microbial

consortia useful for environmental restoration • Developmental genetics, genomics