Chapter 19: Recombinant DNA Technology
19.1 Recombinant DNA Technology combines several techniques
Recombinant DNA is a combination of DNA molecules that are not found in nature. Crossing over creates new combinations of DNA, but they are from the same place. Recombinant is generally used to refer to DNA joined from two different sources. This method is actually a combination of several techniques for producing potentially unlimited quantities of a gene.
The basic procedure:
- DNA to be cloned is purified from cells or tissues
- Restriction enzymes are used to generate specific DNA fragments. These enzymes cut DNA at specific sequences.
- These fragments serve as carrier molecules called vectors. The vector plus the DNA fragment is called the recombinant DNA molecule.
- The recombinant DNA molecule is inserted into a host cell and replicates. This replication produces many identical copies (clones) of the recombinant DNA.
- As the host replicates, the recombinants pass on to the offspring copies of the cloned sequence.
- The cloned DNA is recovered from the host cells, purified, and analyzed.
- The cloned DNA can be transcribed and the mRNA is translated into a product that can be isolated and used for research or sold.
19.2 Recombinant DNA Technology is the foundation of genome analysis
The human genome has more than 3 billion nucleotides and 25000 to 30000 genes. Restriction enzymes can cut the genome into smaller fragments that can be manipulated, separated, copied, and studied individually. This allows researchers to investigate the aspects of gene organization and function.
19.3 Restriction enzymes cut DNA at specific recognition sequences
Hamilton Smith accidentally discovered that Haemophilus influenza, a bacterium, could chop DNA. It holds restrictionenzymes that restrict activity to certain sites on the DNA. In several years, hundreds of bacterial strains were found to hold these enzymes. The enzymes recognize and can cut DNA into readable strands. (Bio 2 – Concepts and Applications)
Restriction enzymes are produced by bacteria as a defense against infection by viruses. More than 200 of these enzymes have been identified, 100 of which were used by researchers. The enzyme binds to DNA at a specific sequence called a recognition sequence. It cuts the strands of DNA within the recognition sequence. These cut pieces are called restriction fragments. The size of the fragments is dependent on how often the enzyme cuts the strand. The usefulness of an enzyme is based on the ability to accurately and reproducibly cut the fragments.
19.4 Vectors carry DNA molecules to be cloned
DNA restriction fragments cannot directly enter a host cell for copying. However, the fragment is joined to another molecule, a vector, and can enter the host cell. Vectors are carrier DNA molecules that can transfer and replicate inserted DNA fragments. Many different vectors are available; each one having different host cell specificities.
Specificities include: differences in the size of inserts that can be carried, differences in number of copies produced, number of recognition sequences available for cloning, and the number and type of marker genes.
Plasmid vectors – derived from double stranded DNA molecules that replicate independently within bacteria.
19.7 Genes can be transferred to eukaryotic cells
Plant and animal cells can take up DNA from their environment. When the vector is a plasmid, DNA transfer is termed transformation. If the vector is a virus, the term transfection is used.
Mammalian cell hosts – DNA can be transferred into mammalian cells by several methods:
Encapsulation of DNA in an artificial membrane that is then fused with the cell membrane
Vectors of retroviruses or a yeast artificial chromosomes
19.8 The polymerase chain reaction makes DNA copies without host cells
In 1986, Kary Mullis developed this method that revolutionized recombinant DNA methodology. This technique eliminates the need for host cells in DNA cloning.
PCR copies a specific DNA sequence through a series of in vitro reactions. Primers are added to a sample of DNA that has been converted into single strands. The primers bind to complementary nucleotides in the sequence to be cloned. DNA polymerase is added to synthesize a second strand of DNA. Repeating these steps makes more copies of the DNA.
Steps:
- DNA is denatured into single strands. (Source can be from anything).Heating to 90-95°C denatures the DNA, causing it to dissociate into single strands. This usually takes 5 minutes.
- The temperature is then lowered to between 50 and 70°C, and at this temperature, primers will bind to the single strand of DNA. The primers serve as starting points for synthesizing new DNA strands complementary to the target DNA.
- DNA polymerase is added to the reaction mixture. DNA synthesis is carried out.
PCR is a chain reaction and the number of new DNA strands is doubled in each cycle; the new strands and old strands serve as templates in the next cycle. Each cycle takes about 5 minutes, so in 25-30 cycles, more than a millionfold increase in DNA.
Limits of PCR – there must be some information about the nucleotide sequences that is known. Minor contamination of the source can cause problems.
Other applications – It quickly identifies restriction enzyme recognition sequences. It can screen for mutations in genomic DNA. It can settle pedigree debates, and help in the ban of whale sale products.
19.9 Libraries are collections of cloned sequences
Genomic libraries contain at least one copy of all of the sequences in an organism’s genome. Libraries are constructed using host cell cloning methods. The DNA is extracted from cells, cut with restriction enzymes, and the fragments are put into vectors.