MANIPULATING DNA:TOOLS &

TECHNIQUES

Nature, Chapter 12

Genetic engineering Scientists who use gene manipulation technology

are sometimes called ‘genetic engineers’. Genetic engineers work with the genetic material,

DNA. Some of the tools and techniques used for gene

manipulation include:Restriction enzymesElectrophoresisProbesLigase enzymeVectorsGene cloning

Restriction enzymes Cut DNA molecules at a particular site All have their own recognition sequence Occur naturally in microorganisms, such

as bacteria Are named according to their source;

eg. EcoRI is the first restriction enzyme found in strain R of the bacterium Escherichia coli.

Are also called cutting enzymes.

Restriction enzymes

Gel electrophoresis Electrophoresis is a technique for sorting

DNA fragments of different lengths. The process involves the following steps:

1. A mixture of DNA fragments is loaded into a ‘well’ in a gel

2. An electrical current is passed through the gel.3. Negatively charged DNA fragments move

towards the positive pole.4. The shorter the fragment of DNA, the quicker it

moves through the gel.

Gel electrophoresis

DNA Profiling DNA profiling is a technique used to identify DNA

from different individuals. Some regions of DNA in human chromosomes

vary in length because of different numbers of repeats of DNA sequences.

The number of repeated sequences varies from one person to another.

Repeated sequences are inherited like other alleles.

The pattern of repeated sequences can be revealed by radioactive or fluorescent probes.

DNA Profiling

DNA Sequencing DNA sequencing is the identification of the exact

order of bases in a DNA molecule. An automated sequencing system:

Uses different coloured fluorescent dyes, each binding to a specific base

Uses a single-stranded copy of DNA as a template for sequencing

Makes complementary copies of the template, with each copy being one nucleotide longer than the previous one

Has data processes by computer and prints the nucleotide sequence.

Recombinant DNA Recombinant DNA is a molecule of DNA

formed from fragments of other DNA molecules.

Steps involved in making recombinant DNA include:Cutting different DNA molecules with the same

restriction enzymeJoining DNA fragments using DNA ligase.

Genes form one species can be combined with those form another species in this manner.

Recombinant DNA

DNA Amplification (PCR) DNA amplification is the production of

potentially millions of copies of a single piece of DNA.

The polymerase chain reaction (PCR) is a method of DNA amplification.

The enzyme used is a heat-resistant DNA polymerase.

The method involves many cycles of denaturing DNA, binding and extending the primers.

DNA Amplification (PCR)

Gene cloning Gene cloning is a method of producing many

copies of a particular piece of DNA. One technique involves inserting a particular

gene into a plasmid. The plasmid with its foreign gene is then

introduced into a bacterial cell. The plasmid multiplies within the bacteria

and reproduces as the bacteria reproduce. Every time the plasmid is replicated, the

gene is replicated.

Gene cloning

Genetic transformation Genetic transformation occurs when DNA from one

species is introduced and maintained in cells of another species.

Some gene can be transferred between different species.

Transgenic organisms have one or more genes in their genomes that have been artificially introduced from another species.

The most commonly modified genetic organisms are crop plants, including corn, wheat, soya beans and canola. Many of these crops have been made wither inset resistant or herbicide resistant.

Gene Delivery Systems: Vectors How a gene is delivered into an

organism depends on the type of organism. Examples are:Organism into which gene is introduced

Gene delivery system (or vector)

Bacteria plasmidbacteriophage

Plant micro-projective bombardmentelectroporationAgrobacterium tumefaciens (a bacterium)

Animal retrovirusmicro-injection

Gene probes A gene probe is a small piece of single-

stranded DNA (or RNA) with a ‘label’ so that it can be located.

The label can be either a radioactive or fluorescent marker.

The base sequence of a gene probe must be complementary to a portion of one of the strands of the DNA target.

A gene probe can locate a particular piece of DNA from among many fragments of DNA.

Gene probes