Recombinant DNA Unit 12 Lesson 2

EcoRI cutting pattern in nucleotide sequence GAATTC RESTRICTION ENZYMES Genetic engineers use restriction enzymes to cut DNA into fragments at or near recognition sites of specific nucleotides known as restriction sites. Naturally occurring restriction enzymes function as a bacterium’s defense mechanism against foreign DNA. Example: EcoRI is a restriction enzyme originally found in E. coli bacteria. It always cuts DNA at nucleotide sequence GAATTC. EcoRI cutting pattern in nucleotide sequence GAATTC

STICKY AND BLUNT ENDS Fragments of DNA that end with unpaired nucleotides have cohesive ends or sticky ends. If vector DNA and target DNA are both cut with the same restriction enzyme, they will both have complementary (A+G or C+T) sticky ends. DNA fragments that end with complete base pairs have blunt ends.

LIGATION In order for DNA fragments to join together, genetic engineers must seal them using an enzyme called DNA ligase. Because they can base-pair with one another, ligation of complementary sticky ends is more efficient than blunt end ligation. DNA composed of fragments from multiple sources is known as recombinant DNA.

TRANSFORMATION Genetically altering a cell with foreign DNA is called transformation. A cell’s ability to alter its genetics by incorporating foreign DNA into its own genome is known as its competence. In order to make cells more permeable to foreign DNA, genetic engineers artificially induce competence by exposing the cell to hostile conditions. Plasmid Vector Transformed Cell

RECOMBINANT DNA RECAP Restriction enzymes cut DNA into fragments Competence is induced and the cell is transformed (genetically altered) Recombinant DNA (DNA fragment + vector DNA) is inserted into a cell’s genome DNA fragments are sealed into vectors using DNA ligase Restriction enzymes cut DNA into fragments

GENOMIC LIBRARIES An organism’s complete set of genetic material is called its genome. Genetic engineers can store an organism’s genome by extracting DNA from its cells, cutting it into fragments using restriction enzymes, inserting the fragments into a population of vectors, and sealing the fragments together with DNA ligase. This collection of vectors is known as a genomic library. DNA is extracted from cells DNA is digested into fragments using restriction enzymes DNA is inserted into vectors and sealed with DNA ligase

POLYMERASE CHAIN REACTION Polymerase Chain Reaction (PCR) is a technique used by molecular biologists to replicate many copies of specific sequences of DNA. The procedure requires primers, DNA polymerase, and nucleotides. Primers are short chains of about 20 nucleotides that are complementary to a region in the target DNA sequence. DNA Replication

STEPS OF PCR Denaturation Annealing Extension The process of PCR involves heating and cooling DNA at regular intervals inside a thermal cycler. Denaturation: DNA is heated to 94°C in order to break down the hydrogen bonds holding its complementary bases together. Annealing: The DNA is cooled to 54°C and primers attach or anneal to its two strands. Extension: The temperature is then raised to 72°C and DNA Polymerase binds to the primers and adds nucleotides.

DNA FINGERPRINTING Restriction fragment length polymorphism (RFLP) is a technique used by molecular biologists to identify individuals based on unique patterns of restriction enzyme cutting in sequences of DNA. RFLP Analysis detects these differences (polymorphisms) through a process involving gel electrophoresis.

GEL ELECTROPHORESIS Gel electrophoresis is a technique used to separate DNA fragments according to their size. Fragments are placed into wells on a sheet of gelatin and an electric current is applied to the sheet. Since DNA is negatively charged, the fragments will migrate toward the positive pole of the sheet. Short fragments travel further from the sample wells than the slower-moving large fragments, creating bands where they accumulate. DNA bands must be stained to make them visible. Marker Sample A Sample B Sample C