1. Manipulating DNA

2. 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

3. 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.

4. 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

5. 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

6. 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

7. 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.

8. 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.

9. 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

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

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

12. 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

13. 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.

14. 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.

15. 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

16. 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.

17. 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

18. 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

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

20. Gel Electrophoresis

21. 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