II. DNA cloning.

III. DNA libraries.

Terms

Erratum Page 296, lines 6-7: should read, "...in the presence of antibiotics."

I. DNA clones are populations of bacteria or yeast that have descended from a single cell into which a piece of foreign DNA was inserted and which replicates with the cell. The procedures that are used to manipulate DNA in bacteria and other organisms are often called genetic engineering. Certain enzymes catalyze reactions with DNA and are essential tools in genetic engineering.

A. Restriction endonucleases (restriction enzymes) break double-stranded DNA at specific nucleotide sequences (restriction sites).
1. The most useful are those that cleave at palindromic sequences. These are DNA sequences that read the same on both strands but in opposite directions. For example, 5'-AGCT-3' on one strand and 3'-TCGA-5' on the complementary strand.

2. The cleavage may produce blunt ends or sticky ends.

3. There are hundreds of known restriction enzymes, each specific for sequences consisting of 4 to 8 nucleotides.

4. The entire DNA of a human would be broken into millions of restriction fragments by some restriction enzymes, but the sequences cleaved would be identical. All the ends of the fragments would be identical also.

B. DNA polymerase uses a single strand template to make a complementary strand. However, it requires a primer and can only add nucleotides to the 3' end of the primer.

C. DNA ligase ties together adjacent nucleotides in a DNA strand if they are bound to the same complementary strand.

D. Reverse transcriptase is a DNA polymerase that uses RNA as a template to make a DNA copy.

A. Preparation of a recombinant DNA vector includes the following steps.
1. Break the vector with a restriction enzyme that cleaves at one point.

2. Use the same restriction enzyme to cleave DNA to be inserted into the vector.

3. Mix the two, allowing the sticky ends to anneal (form hydrogen bonds between complementary strands.)

4. Tie the broken strands together with a ligase.

5. Insert the recombinant plasmid (recombinant DNA) into a host bacterium, which can be cultured to produce any amount of bacteria and plasmids.

B. Yeast artificial chromosomes (YACs) are similar in purpose to bacterial clones, but they can accommodate very much larger pieces of DNA. The constructed chromosomes are inserted into yeast cells and replicate with the normal yeast chromosomes.

C. Some uses of recombinant DNA:

1. Plasmids can be harvested from the bacteria, treated with the same restriction enzyme (or another), and the inserted DNA can be recovered in unlimited quantities.

2. If the inserted DNA is a functional gene, it can be used to produce the corresponding protein. E.g., human insulin, human growth hormone, and human anti-hemophilic factor.

A. Various sources of DNA can be used to create libraries.
1. For a genomic library, one starts with a mixture of DNA fragments obtained by digesting the entire DNA of an organism with a particular restriction enzyme. All the genes in the organism should be represented in the library.

2. Chromosome libraries are made by starting with a DNA preparation from a particular chromosome. This can be done by sorting metaphase chromosomes in a sorting machine that recognizes the small size difference among the chromosomes.

3. Expression libraries are created from the genes that are expressed in a particular tissue. The messenger RNA from that tissue can be isolated by procedures that recognize poly-A tails, DNA copies of the mRNA are then made using an enzyme (reverse transcriptase) that makes DNA copies of RNA templates (called cDNA), and the cDNA is then used as above.

B. In order to select a bacterial or yeast cell that has a piece of DNA of interest, one must use a specific DNA probe.
1. A DNA probe is any single-stranded segment of DNA that is used to detect the presence of a complementary strand by forming a hybrid with it. The probe must be radioactive, fluorescent, or otherwise modified to be detectable.

2. Probes are used to detect positive bacterial colonies as follows:

a. A very dilute suspension of cells is spread on a culture plate so that each of the resulting colonies arises from a single cell.

b. The colonies are transferred to a nitrocellulose or nylon membrane that binds DNA.

c. DNA on the membrane is denatured by heat.

d. DNA on the membrane is flooded with single-stranded ³²P-labeled DNA probe that is allowed to anneal to the membrane-bound DNA.

e. The label is detected by exposure of X-ray film.

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