principles of recombinant dna technology

Biotechnology

Manipulation of organisms for useful products.

Genomic DNA

Long DNA containing introns, used for mapping.

Complementary DNA (cDNA)

Synthesized from mRNA using reverse transcriptase.

Gene Cloning

Inserting DNA fragment into a vector.

Plasmids

Circular DNA replicating independently in bacteria.

Restriction Enzymes

Cut DNA at specific nucleotide sequences.

Transformation

Uptake of DNA by bacterial cells.

Blue-White Screening

Identifies recombinant bacteria using B-galactosidase.

Genomic Library

Collection of clones containing every gene of an organism.

Hybridization Probing

Technique to identify specific DNA sequences in colonies.

Eukaryotic Cells

Cells containing a nucleus and organelles.

Prokaryotic Cells

Simple cells without a nucleus, like bacteria.

Recombinant DNA

DNA molecule formed by combining DNA from different sources.

RNAse H

Degrades mRNA after cDNA synthesis.

DNA Ligase

Enzyme that joins DNA fragments together.

Selectable Marker

Gene allowing identification of transformed cells.

Origin of Replication

Site enabling plasmid replication in host cells.

Competent Cells

Bacteria treated to enhance DNA uptake ability.

Antibiotic Resistance Gene

Gene providing resistance to specific antibiotics.

Sticky Ends

Overhanging ends of DNA after restriction enzyme cuts.

Intron-less Gene

Gene without non-coding sequences, suitable for bacteria.

Colony/Clone

Population of identical cells derived from one cell.

how is cDNA made

synthesised from mrna using reverse transcriptase, requires primer and nucleotides

steps in gene cloning

dna fragment with gene of interest is inserted into circular dna vector, vector transports gene into host - vector multiplies

colony of identical host cells is made

3 important features in plasmids

origin of replication, selectable marker, cloning/restriction enzyme cleavage site

restriction enzyme function

cleave dna at specific nucleotide sequences, leaving sticky ends - leave 5' phosphate and 3'hydroxyl

transformation - shocking process

heat/chemical shocking, bacteria undergo treatment which enhances their ability to take up dna - competent. chemical - soaked in ice cold CaCl solution

issues with recombinant identification

all circular molecules will be cloned including cells containing self-ligated vectors and wrong recombinant molecules

transforming bacteria is called

transformation

transforming eukaryotes is called

transvection

gene libraries

collection of clones sufficient in number to be likely to contain every gene present in a particular organism - hybridisation used to work out which colony contains a gene of interest

What is the first step in hybridisation probing?

Colonies are transferred to a nitrocellulose membrane.

What treatment is applied to the colonies on the nitrocellulose membrane?

They are treated to leave only DNA.

How is DNA attached to the nitrocellulose membrane?

DNA is attached to the membrane through its backbone.

What type of marker is used on the probe in hybridisation probing?

The probe is labelled with a radioactive marker.

What is added to promote nucleic acid hybridisation?

A solution is added to promote nucleic acid hybridisation.

How is the label detected in hybridisation probing?

The label is detected to identify colonies.