Download the VECTOR (gene carrier)

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DNA TECHNOLOGY- methods for studying and manipulating genetic material.
BIOTECHNOLOGY, the manipulation of organisms or their components to make useful
products. Biotechnology today usually refers to DNA technology, modern laboratory
techniques that involve the manipulation of DNA.
RECOMBINANT DNA is formed when scientists combine nucleotide sequences from two
different sources-often different species- to form a single DNA molecule. Scientists have
genetically engineered bacteria to mass-produce a variety of useful chemicals, from cancer
drugs to pesticides.
To manipulate genes in a laboratory, biologist often use bacterial PLASMIDS, which are
small, circular DNA molecules that replicate separately from the much larger bacterial
chromosome. GENE CLONING, the production of multiple identical copies of a genecarrying piece of DNA. Gene-cloning methods are central to GENETIC ENGINEERING, the
branch of biotechnology that involves the direct manipulation of genes for practical
purposes.
To begin, the biologist isolates two kinds of DNA: 1.) A bacterial plasmid that will serve as
the VECTOR (gene carrier)
2.) The DNA containing gene is isolated. The researcher treats both the plasmid and the
gene with an enzyme that cuts each DNA.
3.) An enzyme is chosen that cleaves the plasmid in only one place.
4.) The other DNA, which is usually much longer in sequence, may be cut into many
fragments. The cuts leave single stranded ends.
5.) The cut DNA from both sources (plasmid and target) are mixed. The single-stranded
ends of the plasmid base-pair with the complementary ends of the target DNA fragment.
6.) The enzyme DNA LIGASE joins the two DNA molecules by covalent bonds. This enzyme,
which the cell normally uses in DNA replication is a DNA pasting enzyme that catalyzes the
formation of covalent bonds between adjacent nucleotides, joining the strands. The result
is a recombinant DNA plasmid.
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7.) The recombinant plasmid containing the target gene is mixed with a culture of bacteria.
Under the right conditions a bacterium takes up the plasmid DNA by transformation.
8.) This is recombinant bacterium then reproduces to form a CLONE of cells (a group of
identical cells descended from a single ancestral cell), each carrying a copy gene.
9.) Genes or proteins are isolated from the cloned bacterium.
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The cutting tools used are bacterial enzymes called RESTRICTION ENZYMES. The DNA
sequence recognized by a particular restriction enzyme is called a RESTRICTION SITE. Once
a restriction site is recognized, the restriction enzyme cuts both strands of the DNA at
specific points within the sequence.
1.) We start with a piece of DNA containing one recognition sequence for the restriction
enzyme EcoRI. The restriction enzyme cuts the DNA strands between the bases A and G
within the sequence, producing pieces of DNA called RESTRICTION FRAGMENTS.
2.)The staggered cuts yield two double-stranded DNA fragments with single-stranded ends,
called “sticky ends”. Sticky ends are the key to joining DNA restriction fragments originating
from different sources.
3.) A piece of DNA from another source is now added. The foreign DNA has ends with this
particular base sequence because it was cut from a large molecule by the same restriction
enzyme used to cut the DNA.
4.) The complementary ends both fragments allow them to stick together by base-pairing.
The union of complementary bas-pairing is made permanent by the pasting enzyme DNA
LIGASE.
5.) The final outcome is a stable molecule of recombinant DNA.
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A GENOMIC LIBRARY is the entire collection of all the cloned DNA fragments from a
genome.
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1.) The chosen cells transcribe their genes.
2.) Process the transcripts to produce mRNA.
3.) The researcher isolates the mRNA and makes single-stranded DNA transcripts from it
using the enzyme REVERSE TRANSCRIPTASE, which is obtained from retroviruses.
4.) Another enzyme is added to break down the mRNA.
5.) DNA polymerase is used to synthesize a second DNA strand.
The DNA that results from such a procedure, called COMPLEMENTARY DNA (cDNA),
represents only the subset of genes that had been transcribed into mRNA in the starting
cells.
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Methods for detecting genes directly depend on base pairing between the gene and a
complementary sequence on another nucleic acid molecule, either DNA or RNA. NUCLEIC
ACID PROBES are used to find a specific gene or other nucleotide sequence within a mss of
DNA.
First, the DNA sample is treated with heat or alkali to separate the DNA strands. When the
DNA probe is added to these strands, it tags the correct molecule- finds the correct shelf in
the library- by hydrogen-bonding to the complementary sequence in the gene of interest.
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