Manipulating DNA

Manipulating DNA Scientists use their knowledge of the structure of DNA and its chemical properties to study and change DNA molecules Different techniques are used to study and change DNA molecules Genetic Engineering – making changes in the DNA code of a living organism Bacteria are the workhorses of modern biotechnology

Tools of Molecular Biology: Recombinant DNA Recombinant DNA techniques can help biologists produce large quantities of a desired protein To work with genes in the laboratory, biologists often use bacterial plasmids, small, circular DNA molecules Plasmids can: can carry virtually any gene, can act as vectors, DNA carriers that move genes from one cell to another, and are ideal for gene cloning, the production of multiple identical copies of a gene-carrying piece of DNA.

Recombinant DNA Recombinant DNA is produced by combining two ingredients: a bacterial plasmid and the gene of interest. To combine these ingredients, a piece of DNA must be spliced into a plasmid

Creating Recombinant DNA In order to create Recombinant DNA, there needs to be: DNA extraction Cells opened to separate DNA from other cell parts Cutting DNA DNA too large to study, so biologists “cut” them into smaller fragments using restriction enzymes. Many restriction enzymes are known and each one cuts DNA at a specific sequence of nucleotides Produces pieces of DNA called restriction fragments with “sticky ends” important for joining DNA from different sources. Splicing DNA back together DNA ligase connects the DNA pieces into continuous strands by forming bonds between adjacent nucleotides

Restriction Enzymes Recognition site (recognition sequence) for a restriction enzyme Restriction Enzymes 1 A restriction enzyme cuts the DNA into fragments. Restriction enzyme Sticky end Sticky end

Restriction Enzymes Recognition site (recognition sequence) for a restriction enzyme Restriction Enzymes DNA 1 A restriction enzyme cuts the DNA into fragments. Restriction enzyme Sticky end Sticky end 2 A DNA fragment is added from another source.

Restriction Enzymes Recognition site (recognition sequence) for a restriction enzyme Restriction Enzymes DNA 1 A restriction enzyme cuts the DNA into fragments. Restriction enzyme Sticky end Sticky end 2 A DNA fragment is added from another source. 3 Fragments stick together by base pairing.

Restriction Enzymes Recognition site (recognition sequence) for a restriction enzyme Restriction Enzymes DNA 1 A restriction enzyme cuts the DNA into fragments. Restriction enzyme Sticky end Sticky end 2 A DNA fragment is added from another source. 3 Fragments stick together by base pairing. 4 DNA ligase joins the fragments into strands. DNA ligase Recombinant DNA molecule

Recognition sequences Restriction Enzymes Recognition sequences DNA sequence Restriction enzyme EcoRI cuts the DNA into fragments. Sticky end

Recognition sequences Restriction Enzymes Recognition sequences DNA sequence Restriction enzyme EcoRI cuts the DNA into fragments. Sticky end

Finding the Gene of Interest How can a researcher obtain DNA that encodes a particular gene of interest? First, you have to have an idea of what the gene is you want to work with (get a genomic library). Then: Using a nucleic acid probe consisting of a short single strand of DNA with a complementary sequence and labeled with either a radioactive isotope or a fluorescent dye. Or by synthesizing it through reverse transcriptase (viral enzyme that makes DNA) Or by making it by scratch with machines

DNA Profiling and Forensic Science can be used to determine if two samples of genetic material are from a particular individual and has rapidly revolutionized the field of forensics, the scientific analysis of evidence from crime scenes. To produce a DNA profile, scientists compare sequences in the genome that vary from person to person.

DNA Profiling and Forensic Science DNA profiling can be used to test the guilt of suspected criminals, identify tissue samples of victims, resolve paternity cases, identify contraband animal products, and trace the evolutionary history of organisms.

DNA Profiling Techniques: Making Copies of DNA Polymerase Chain Reaction (PCR) technique Allows biologists to make many copies of a specific piece of DNA DNA strands separated with heat, then cooled to allow DNA Polymerase to start making new copies of DNA A few dozen heat and cool cycles results in many copies of DNA

Making Copies of DNA DNA polymerase adds complementary strand DNA heated to separate strands DNA fragment to be copied PCR cycles 1 DNA copies 1 2 3 4 4 8 5 etc. 16 etc.

DNA Profiling Techniques: STR Analysis Short tandem repeats (STRs) are: short sequences of DNA that are repeated many times, tandemly (one after another), in the genome. STR analysis Proves two samples come from the same person - Everyone has these repetitive DNA sequences, but in different lengths and a different number of them compares the lengths of STR sequences at specific sites in the genome uses gel electrophoresis, a method for sorting DNA by size

DNA Profiling Techniques: Gel Electrophoresis Used to separate DNA fragments. DNA fragments placed in a gel and electricity is applied to the gel. DNA molecules are negatively charged and move towards the positive end of the gel. Smaller DNA fragments move faster and farther This technique used to compare the genomes of different organisms or even different people

Gel Electrophoresis Power source DNA plus restriction enzyme Longer fragments Shorter fragments Mixture of DNA fragments Gel

Gel Electrophoresis

Blood found at Crime Scene Gel Electrophoresis Which suspect should have more questioning? DNA from Victim DNA from Suspect #1 DNA from Suspect #2 DNA from Suspect #3 DNA from Suspect #4 Blood found at Crime Scene