NIS - BIOLOGY Lecture 57 – Lecture 58 DNA Technology Ozgur Unal 1

Genetic Engineering 2  What comes to your mind when you hear the term “Genetic Engineering”?

Genetic Engineering 3  Genetic engineering is the technology that involves manipulating the DNA of one organism in order to insert exogenous DNA (DNA of another organism).  Example: Researchers have inserted a gene for a bioluminescent protein called green fluorescent protein (GFP) into various organisms.  GFP is naturally present in jellyfishes in the north Pacific Ocean  Some organisms have been genetically engineered to synthesize GFP.  Figure 13.3  mosquito larvae

Genetic Engineering 4  Selective breeding is used to produce plants and animals with desired traits.  Similarly, genetic engineering can be used to increase or decrease the expression of specific genes in selected organisms.  Genetic engineering has many applications  human health, agriculture etc.  But how are these engineering processes achieved?

DNA Tools 5  An organism’s genome is the total DNA present in the nucleus of each cell.  Human genome contains around 25,000 thousand genes.  In order to study a specific gene, DNA tools can be used to manipulate DNA and to isolate genes from the rest of the genome.

DNA Tools 6 Restriction Enzymes:  Some types of bacteria contain powerful defenses against viruses.  These cells contain proteins called restriction enzymes that recognize and bind to specific DNA sequences and cleave the DNA within that sequence.  A restriction enzyme, also called endonuclease, cuts the viral DNA into fragments after it enters the bacteria.  There are many different types of restriction enzymes.

DNA Tools 7 Restriction Enzymes: EcoRI  One restriction enzyme that is widely used by scientists is EcoRI.  EcoRI specifically cuts DNA containing the sequence GAATTC.  EcoRI cuts this sequence such that it produces complementary sticky ends.  Not all restriction enzymes create sticky ends! 

DNA Tools 8 Gel Electrophoresis:  An electric current is used to separate the DNA fragments according to the size of the fragments in a process called gel electrophoresis.  Check out Figure 13.5!!  When an electric current is applied, the DNA fragments move toward the positive end of the gel.  The smaller fragments move faster than the larger ones.  Portions of the gel containing each band can be removed for further study.

Recombinant DNA Technology 9  After DNA fragments have been separated by gel electrophoresis, fragments can be removed and combined with DNA fragments from another source  recombinant DNA  Large quantities of recombinant DNA are needed to study them  In order to transfer the recombinant DNA into a bacterial cell, scientists use a carrier (also called vector)  Plasmid or Virus  Plasmids are small, circular and double stranded DNA molecules that occure naturally in bacteria and yeast.

Recombinant DNA Technology 10  If a plasmid and a DNA fragment obtained from another genome have been cleaved by the same restriction enzyme, the ends of each DNA fragment will be complementary and can be combined.  An enzyme normally used by cells in DNA repair and replication, called DNA ligase, joins the two DNA fragments chemically.  Check out Figure 13.6!!

Gene Cloning 11  Gene cloning is used to make large quantities of recombinant plasmid DNA.  Some bacterial cells take up the recombinant plasmid DNA into them through a process called transformation.  Bacteria can be transformed using electric pulsation or heat.  Large quantities of identical bacteria, each containing the inserted DNA molecules, can be produced through a process called cloning.  Check out Figure 13.7!!

DNA Sequencing 12  The sequence of the DNA nucleotides of most organisms is unknown.  Knowing the sequence is a valuable is information to have.  Check out Figure 13.8 for the steps in DNA sequencing technique!!

Polymerase Chain Reaction 13  Once the sequence of a DNA fragment is known, a technique called the polymerase chain reaction, PCR, can be used to make millions of copies of a specific region of a DNA fragment.  PCR is extremely sensitive and can detect a single DNA molecule in a sample.  Follow the steps of PCR from the book and check out Figure 13.9!!  Check out Table 13.1 for the differences in the DNA manipulation techniques we learned so far.