DNA Cloning - Fig 20.1
To study a particular gene, you need to isolate only the small, well-defined, portion of a chromosome containing the gene.
Gene cloning makes multiple identical copies of gene-sized pieces of DNA.

A foreign gene is inserted into a bacterial plasmid and this recombinant DNA molecule is returned to a bacterial cell.
Every time this cell reproduces, the recombinant plasmid is replicated as well and passed on to its descendents.
Under suitable conditions, the bacterial clone will make the protein encoded by the foreign gene.
Uses of cloned genes:
    To produce a protein product, like human growth hormone.
    To prepare many copies of the gene itself.
        to determine the gene's nucleotide sequence

        to provide an organism with a new metabolic capability by transferring a gene from another organism.

Restriction enzymes are used to make recombinant DNA - Fig 20.2.

Restriction enzymes cut DNA molecules at specific locations called restriction sites.
In nature, bacteria use restriction enzymes to cut foreign DNA and protect their own DNA by methylation.
Restrictions enzymes recognize short DNA nucleotide sequences and cut at specific point in these sequences.
Because the target sequence usually occurs many times on a long DNA molecule, an enzyme will make many cuts.
Copies of a DNA molecule will always yield the same set of restriction fragments when exposed to a specific enzyme.
Restriction enzymes leave sticky ends.
These will bond with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme.
DNA ligase seals the strand.

Genes can be cloned in recombinant DNA vectors - Fig 20.3.

Recombinant plasmids are produced by splicing restriction fragments from foreign DNA into plasmids.
The original plasmid is called a cloning vector.

The process of cloning a human gene in a bacterial plasmid can be divided into five steps.

   1) Isolation of vector and gene-source DNA.

The source DNA comes from human tissue cells.
The source of the plasmid is typically E. coli.
This plasmid carries two useful genes, ampR, conferring resistance to the antibiotic ampicillin and lacZ, encoding the enzyme beta-galactosidase which catalyzes the hydrolysis of sugar.

   2) Insertion of DNA into the vector.

same restriction enzyme

sticky ends
DNA ligase.·

   3) Introduction of the cloning vector into cells.

Bacterial cells that are are lacZ- take up the recombinant plasmids by transformation.
Some bacteria have taken up the desired recombinant plasmid DNA, other bacteria that have taken up other DNA.

   4) Cloning of cells (and foreign genes).

Plate out the transformed bacteria on a solid nutrient medium containing ampicillin and a sugar called X-gal.
Only bacteria that have the ampicillin-resistance plasmid will grow.

The X-gal in the medium is used to identify plasmids that carry foreign DNA.
Bacteria with plasmids lacking foreign DNA stain blue when beta-galactosidase hydrolyzes X-gal.
Bacteria with plasmids containing foreign DNA are white because they lack beta-galactosidase.

   5) Identifying cell clones with the right gene.

Sort through the bacterial colonies with foreign DNA to find those containing the gene of interest.
Nucleic acid hybridization, depends on base-pairing between the gene and a complementary sequence, a nucleic acid probe, on another nucleic acid molecule. Fig 20.4.
A radioactive or fluorescent tag labels the probe.
The probe will hydrogen-bond specifically to complementary single strands of the desired gene.
After denaturation (separating) the DNA strands in the plasmid, the probe will bind with its complementary sequence, tagging colonies with the targeted gene.

Inducing a cloned eukaryotic gene to function in a prokaryotic host can be difficult so employ an expression vector, a cloning vector containing the requisite prokaryotic promotor upstream of the restriction site.

The presence of introns creates problems for expressing these genes in bacteria. Fig. 20.5.

To express eukaryotic genes in bacteria, a fully processed mRNA acts as the template for the synthesis of a complementary strand using reverse transcriptase.
This complementary DNA (cDNA), with a promoter, can be attached to a vector for replication, transcription, and translation inside bacteria.

Can use eukaryotic cells as host for cloning and expressing eukaryotic genes

Yeast cells, single-celled fungi, are as easy to grow as bacteria and have plasmids, rare for eukaryotes.
Scientists have constructed yeast artificial chromosomes (YACs) - an origin site for replication, a centromere, and two telomeres -with foreign DNA.
These chromosomes behave normally in mitosis and can carry more DNA than a plasmid.
Eukaryotic hosts are capable of providing the posttranslational modifications that many proteins require.

Several techniques facilitate entry of foreign DNA.

electroporation
microscopically thin needles.
For plants, DNA is attached to microscopic metal particles and fired into cells with a gun.

Cloned genes are stored in DNA libraries - Fig 20.6

A complete set of recombinant plasmid clones, each carrying copies of a particular segment from the initial genome, forms a genomic library.
The library can be saved and used as a source of other genes or for gene mapping.
Certain bacteriophages are also common cloning vectors for making libraries.
    Fragments of foreign DNA can be spliced into a phage genome using a restriction enzyme and DNA ligase.
    The recombinant phage DNA is packaged in a capsid in vitro and allowed to infect a bacterial cell.
    Infected bacteria produce new phage particles, each with the foreign DNA.

The polymerase chain reaction (PCR) clones DNA entirely in vitro - Fig. 20.7

DNA cloning is the best method for preparing large quantities of a particular gene or other DNA sequence.
When the source of DNA is scanty or impure, the polymerase chain reaction (PCR) is quicker and more selective.
The DNA is incubated in a test tube with special DNA polymerase, isolated from bacteria living in hot springs, a supply of nucleotides, and short pieces of single-stranded DNA as a primer.
PCR can make billions of copies of a targeted DNA segment in a few hours, much faster than cloning with recombinant bacteria.
In PCR, a three-step cycle-heating, cooling, and replication-brings about a chain reaction that produces an exponentially growing population of DNA molecules.

Examples:
   Fragments of ancient DNA from a 40,000-year-old frozen woolly mammoth.
   DNA from tiny amount of blood or semen found at the scenes of violent crimes.
   DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders.
   DNA of viral genes from cells infected with difficult-to-detect viruses such as HIV.

DNA Analysis and Genomics

Comparisons among whole sets of genes and their interactions is the field of genomics.
One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis. Fig 20.8.
Gel electrophoresis separates macromolecules - nucleic acids or proteins - on the basis of their rate of movement through a gel in an electrical field.
Rate of movement depends on size, electrical charge, and other physical properties of the macromolecules.
For linear DNA molecules, separation depends mainly on size, with longer fragments migrating less along the gel.

Restriction fragment analysis detects DNA differences that affect restriction sites - Fig 20.9.

After treating long DNA molecules with a restriction enzyme, the fragments can be separated by size via gel electrophoresis.
This produces a series of bands that are characteristic of the starting molecule and that restriction enzyme.
The separated fragments can be recovered undamaged from gels, providing pure samples of individual fragments.
We can use restriction fragment analysis to compare two different DNA molecules representing, for example, different alleles, which may differ in one or more restriction sites.
If they do differ in restriction sites, each will produce different-sized fragments when digested by the same restriction enzyme.

The restriction fragments from the two alleles will produce different band patterns.
Restriction fragment analysis is sensitive enough to distinguish between two alleles of a gene that differ by only base pair in a restriction site.

Although electrophoresis will yield too many bands to distinguish individually, we can use nucleic acid hybridization with a specific probe to label discrete bands. The radioactive label on the single-stranded probe can be detected by autoradiography.

To compare 3 individuals:

Add the restriction enzyme to each of the three samples to produce restriction fragments.
Separate the fragments by gel electrophoresis.
Southern blotting (Southern hybridization) allows us to transfer the DNA fragments from the gel to a sheet of nitrocellulose paper, still separated by size. Fig 20.10.
This also denatures the DNA fragments.
Bathing this sheet in a solution containing the probe allows the probe to attach by base-pairing (hybridize) to the DNA sequence of interest and then visualize bands containing the label with autoradiography.
For our three individuals, the results of these steps show that individual III has a different restriction pattern than individuals I or II.
Southern blotting can be used to examine differences in noncoding DNA as well.

Differences in DNA sequence on homologous chromosomes that produce different restriction fragment patterns are called restriction fragment length polymorphisms (RFLPs) and can serve as a genetic marker for a particular location (locus) in the genome.