Lesson 12 Genetics Molecular Genetic Analysis and Biotech

A set of molecular techniques for

Locating, isolating, amplifying, and manipulating DNA fragments

Restriction enzymes

Recognize and make double stranded cuts in DNA at specific sequences called recognition sites.

Gel electrophoresis

DNA is negatively charged and fragments go through the gel

Amplifying DNA fragments with the polymerase chain reaction (PCR)

DNA fragments from one cell is so tiny and needs to be amplfiied.

Two essential components of PCR

Template DNA can be synthesized

A pair of primers which new nucleotides can be added

High temp DNA polymerase

PCR steps

Denature

Primer annealing

Primer extension

What is needed to clone genes?

Cloning vector carrying the foreign DNA piece. Host bacterial cells to host and amplify the vector

Cloning vector

Stable, replicating DNA molecule

Origin of replication

Selectable marker

Unique restriction enzyme sites

Basic steps in Gene cloning?

1. The vector acts as a vehicle that transports the gene into a host cell, usually a bacterium

2. Within the host cell, the vector multiplies, producing numerous identical copies of itself and the inserted gene.

3. When the host cell divides, vector replication takes place, copies of the recombinant DNA molecules are passed to the progeny.

4. After a large number of cell divisions, a clone of identical host cells is produced.

   1. Clones can be detected with selectable markers on plasmid. Use XGAL to identify