Ch. 13 Sec. 2 Manipulating DNA
Scientists use their knowledge of the structure of DNA and its chemical properties to study and change DNA molecules. • extract DNA from cells • cut DNA into smaller pieces • identify the sequence of bases in a DNA molecule • make unlimited copies of DNA
I. The Tools of Molecular Biology A. Genetic engineering - making changes in the DNA code of a living organism • B. DNA Extraction • DNA can be extracted from most cells by a simple chemical procedure. • The cells are opened and the DNA is separated from the other cell parts.
C. Cutting DNA • Most DNA molecules are too large to be analyzed, so biologists cut them into smaller fragments using restriction enzymes.
2. Each restriction enzyme cuts DNA at a specific sequence of nucleotides 3. A restriction enzyme will cut a DNA sequence only if it matches the sequence precisely Recognition sequences DNA sequence Restriction enzyme EcoR I cuts the DNA into fragments Sticky end
Fig. 13-5 Page 322 Molecular biologists have developed different techniques that allow them to study and change DNA molecules. Restriction enzymes cut DNA at specific sequences. This drawing shows how restriction enzymes are used to edit DNA. The restriction enzyme EcoR I, for example, finds the sequence CTTAAG on DNA. Then, the enzyme cuts the molecule at each occurrence of CTTAAG. The cut ends are called sticky ends because they may “stick” to complementary base sequences by means of hydrogen bonds. Recognition sequences DNA sequence Restriction enzyme EcoR I cuts the DNA into fragments Sticky end
D. Separating DNA • Gel electrophoresis • DNA fragments placed in a porous gel • Electric voltage is applied • Negatively-charged DNA molecules move toward the positive end of the gel • Compare the genomes of different organisms • Locate and identify one particular gene
Fig. 13-6 Page 323 Gel electrophoresis is used to separate DNA fragments. First, restriction enzymes cut DNA into fragments. The DNA fragments are then poured into wells on a gel, which is similar to a thick piece of gelatin. An electric voltage moves the DNA fragments across the gel. Because longer fragments of DNA move through the gel more slowly, they do not migrate as far across the gel as shorter fragments of DNA. Based on size, the DNA fragments make a pattern of bands on the gel. These bands can then be compared with other samples of DNA. Power source DNA plus restriction enzyme Longer fragments Shorter fragments Mixture of DNA fragments Gel Gel Electrophoresis
DNA plus restriction enzyme • First, restriction enzymes cut DNA into fragments. • The DNA fragments are poured into wells on a gel. Mixture of DNA fragments Gel
Power source • An electric voltage is applied to the gel. This moves the DNA fragments across the gel. • The smaller the DNA fragment, the faster and farther it will move across the gel.
Based on size, the DNA fragments make a pattern of bands on the gel. • These bands can then be compared with other samples of DNA. Longer fragments Shorter fragments Gel Electrophoresis
II. Using the DNA Sequence A. Reading the Sequence • In DNA sequencing, a complementary DNA strand is made using a small proportion of fluorescently labeled nucleotides
2. Each time a labeled nucleotide is added, it stops the process of replication, producing a short color-coded DNA fragment 3. When the mixture of fragments is separated on a gel, the DNA sequence can be read
Fig 13-7 Page 324 Knowing the sequence of an organism’s DNA allows researchers to study specific genes. In DNA sequencing, a complementary DNA strand is made using a small proportion of fluorescently labeled nucleotides. Each time a labeled nucleotide is added, it stops the process of replication, producing a short color-coded DNA fragment. When the mixture of fragments is separated on a gel, the DNA sequence can be read directly from the gel.
Base sequence as “read” from the order of the dye bands on the gel from bottom to top: T G C A C Electrophoresis gel
B. Cutting and Pasting • DNA synthesizers • Short DNA sequences assembled • Can then be joined to “natural” sequences using enzymes that splice DNA together • Recombinant DNA - a gene from one organism attached to the DNA of another organism
C. Making Copies • Polymerase chain reaction (PCR) • A biologist adds short pieces of DNA (primers) that are complementary to portions of the sequence to be copied • DNA is heated to separate its two strands, then cooled to allow the primers to bind to single-stranded DNA • DNA polymerase starts making copies of the region between the primers
Fig. 13-8 Page 325 Polymerase Chain Reaction (PCR) DNA heated to separate strands DNA polymerase adds complementary strand DNA fragment to be copied PCR cycles 1 2 3 4 5 etc. DNA copies 1 2 4 8 16 etc.
13–2 Manipulating DNA To increase variation, scientists can make changes directly to DNA. Genetic engineering is the intentional changing of an organism’s DNA. Scientist use their knowledge of the structure of DNA and its chemical properties to study and change DNA molecules. • Extracting DNA. Scientists can extract, or separate, DNA from the other cell parts using a chemical procedure. • Cutting DNA. Scientists can cut DNA into smaller pieces using restriction enzymes. • Separating DNA. Scientists use gel electrophoresis, a method in which DNA fragments are put at one end of a porous gel. When an electric current is applied to the gel, DNAmolecules move toward the positive end of the gel. This technique allows scientists to compare the gene composition of different organisms or different individuals.
• Separating DNA. Scientists use gel electrophoresis, a method in which DNA fragments are put at one end of a porous gel. When an electric current is applied to the gel, DNAmolecules move toward the positive end of the gel. This technique allows scientists to compare the gene composition of different organisms or different individuals. Scientists also use different techniques to read, change, and copy the DNA sequence. • Scientists can read the order of nucleotide bases in a DNA fragment. They make a copy of a single strand of DNA with colored nucleotides inserted at random places. Reading the order of colored bands in a gel gives the nucleotide sequence of the DNA fragment.
• Scientists can change DNA sequences. Short sequences of DNA made in the laboratory can be joined to the DNA molecule of an organism. DNA from one organism can be attached to the DNA of another organism. These DNA molecules are called recombinant DNA because combiningDNA from different sources makes them.• Scientists often need many copies of a certain gene to studyc it. A technique called polymerase chain reaction (PCR) allows scientists to do that. PCR is a chain reaction in which DNA copies become templates to make more DNA copies.