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Geospatial Technology

Technology used for visualization,

measurement, and analysis of features or phenomena that occur on the earth

Molecular Techniques
Objectives
Understand the concepts of amplification, hybridization, and

separation as they pertain to the study of nucleic acids.


Know what restriction endonucleases are and understand

how they recognize DNA, how they work, and how they are used by molecular geneticists.
Know what a vector is, and what basic properties it has.

e able to follow the production of a recombinant DNA molecule, from restriction digestion to insertion to library screening.

Understand what polymerase chain reaction is and how it

works.
Understand gel electrophoresis and how it is used. Understand the processes of restriction mapping and

DNA se!uencing.

Basic reasons for using molecular techniques


Amplification Separation and Detection Expression

Techniques
i. ii. iii. iv. v.

Formation of Recombinant DNA Polymerase Chain Reaction Gel electrophoresis DNA Sequencing !pression of Clone" Genes

Formation of Recombinant DNA

"n bacteria, this activity is used to protect bacteria from invasion by bacteriophages # when the bacteriophage in$ects its DNA, the enzymes cut the DNA and prevent replication of the phage. At least, that%s the idea& the strategy isn%t always successful.

'estriction enzymes are named according to the species and strain of bacteria they are isolated from.

(he first letter of the name comes from the genus of the bacterium, the ne)t two letters come from the species, and any subse!uent designations are the strain of bacterium and the order in which the enzyme was discovered in that bacterium.

*or e)ample, the enzyme +co'" was discovered in Eschericia coli, strain ',-., and it was the first restriction enzyme identified in that bacterium /hence the designation "0.

(he polymerase chain reaction is an e)tremely versatile techni!ue for copying DNA.

Polymerase Chain Reaction

"n brief, 12' allows a single DNA se!uence to be copied /millions of times0, or altered in predetermined ways.

12' can be used to introduce restriction enzyme sites, or to mutate /change0 particular bases of DNA, the latter is a method referred to as 34uick change3.

12' can also be used to determine whether a particular DNA fragment is found in a cDNA library.

12' has many variations, like reverse transcription 12' /'(#12'0 for amplification of 'NA, and, more recently, real#time 12' /412'0 which allow for !uantitative measurement of DNA or 'NA molecules.

Gel electrophoresis

5el electrophoresis is one of the principal tools of molecular biology.

(he basic principle is that DNA, 'NA, and proteins can all be separated by means of an electric field.

"n agarose gel electrophoresis, DNA and 'NA can be separated on the basis of size by running the DNA through an agarose gel.

1roteins can be separated on the basis of size by using an 6D6# 1A5+ gel, or on the basis of size and their electric charge by using what is known as a 7D gel electrophoresis.

DNA

equencing

Utilizes a modified DNA replication reaction. As with other

DNA replication reactions, this one requires a primer.


For DNA sequencing, the primer is often complementary to a

sequence within the vector, close to where the DNA of interest is inserted.
To visualize the synthesized DNA, the primer is la eled,

either radioactively or with a fluorescent tag.


!ther components of the reaction are normal components of

a DNA replication reaction, e"cept for one.

!"pression of Clone# Genes "n this techni!ue, DNA coding for a protein of interest is cloned /using 12'
and8or restriction enzymes0 into a plasmid /known as an e)pression vector0. (his plasmid may have special promoter elements to drive production of the protein of interest, and may also have antibiotic resistance markers to help follow the plasmid.

(his plasmid can be inserted into either bacterial or animal cells. "ntroducing DNA into bacterial cells can be done by transformation /via uptake of naked DNA0, con$ugation /via cell#cell contact0 or by transduction /via viral vector0.

"ntroducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection.