1. Chapter 12- DNA Technology and the Human Genome
PCR
Plasmid
Probe
R plasmid
Recombinant DNA
Restriction enzymes
Restriction fragments
Sex pili
Sticky ends
Telomeres
Ti plasmid
Transduction
Transformation
Transgenic organism
Transposons
Vaccine
Vector
Biotechnology
Blunt ends
cDNA
Conjugation
DNA fingerprinting
DNA ligase
DNA microarrays
F factor
Gel electrophoresis
Gene cloning
Gene therapy
Genetic marker
Genomic library
Genomics
GM organisms
Golden rice
Human Genome Project

2. Bacterial mating shows how genes are transferred
Asexual reproduction
3 ways to transfer DNA:
1-Transformation- DNA is taken in from surrounding fluid
Explains Griffiths experiment
2-Transduction- phage transfers bacterial genes
Has genes from previous host

3. Conjugation- cells join and a copy of DNA is transferred via mating bridge
Figures 12.1 A,B, and C in text pg 232

4. Plasmids in conjugation
Small, circular piece of DNA separate from bacterial chromosome
Carry genes during conjugation
F-factor- piece of DNA that carries genes for sex pilli and other genes necessary for conjugation
Behaves 2 ways during conjugation:
1- it is integrated into bacterial chromosome
2- it is a plasmid
When it carries genes for more than replication and conjugation it is acting as a vector
R –plasmids- “resistance” – carry genes for enzymes that destroy antibiotics
They can also carry genes of interest and that gene can then be transcribed and translated

5. Plasmids in conjugation

7. What is biotechnology? Using organisms to perform practical tasks
Bacteria making human proteins (insulin, HGH)
Genes for pest resistance put into plants
Bacteria with genes to break down toxic waste
Golden Rice

8. How is this transformation done?
Restriction enzymes- recognize short nucleotide sequences and cut at a specific point
Used in nature to chop of intruder DNA to protect
Cuts ends in two ways: sticky or blunt
DNA ligase- catalyzes covalent bonds between nucleotides
Putting genes into a piece of DNA using restriction enzymes and DNA ligase is producing recombinant DNA
Genes can be cloned using this method

9. Gene Cloning Produces multiple copies of a gene
Human DNA and bacterial plasmid are cut with same restriction enzyme
They are mixed and with DNA ligase are recombined
The plasmid is put back into the bacteria and allowed to reproduce asexually

10. Uses for recombinant DNA?
Mass produce gene products
“pharm” animals can produce human genes
Human insulin can be produced by E. coli
Certain proteins can now be produced and secreted in an animals milk
Rice fortified with certain vitamins

11. GMO’s genetically modified organisms
Organism that has artificially gained 1 or more genes
To: delay ripening, resistance to spoiling, disease, herbicide, insecticide, add nutrients *Golden rice
Vectors are usually used to introduce genes to plants (fig 12.18A)
Ti plasmid- from soil bacteria (induces tumors)
Researchers have removed tumor causing properties but kept ability to transfer DNA
Recombinant organism is genes of another species is a transgenic organism

13. Keeping track of these pieces
Genomic library-
Set of DNA segments, carried on a plasmid or phage, from an organisms’ genome

14. Making cDNA (complementary)
Using reverse transcriptase-
produces DNA from RNA template
DNA without introns
can be transcribed and translated by bacteria
Bacteria can then make human protein

15. Using probes Short labeled nucleic acid molecule
Used to identify specific gene or DNA sequence
Bacterial colonies are transferred to filter paper
DNA is heated to separate strands
Radioactive probes are added, filter is “developed”
Compare “picture” with culture to find genes

17. DNA microarrays
Used to detect the expression of 1000’s of genes at a time
Tiny pieces of DNA are attached to glass and tested for hybridization with various DNA molecules

18. Gel Electrophoresis Sorts molecules by size and charge
Samples are added to wells in a gel
Electric current allows molecule movement
Small molecules move farther and faster

23. Genetic Markers and probes are used with gel electrophoresis
Genetic markers- chromosomal landmarks that can be used to study inheritance
Non-coding sequences- similar in related individuals
DNA is cut with restriction enzymes, and run through electrophoresis gel
Probes can then be used to detect specific sequences

25. What if the sample of DNA we have it too small to run a gel?
PCR-
polymerase chain reaction
amplifies amount of DNA in test tube without using living cells
DNA sample is put into tube with DNA pol, nucleotides, and other molecules (primers)
Sample is heated to separate strands then cooled for nucleotides to bind (done over and over again)

28. Uses for gel electrophoresis and PCR?
DNA Fingerprinting- uses short repeats and PCR to amplify

29. Ironically… Most of your DNA is junk (97%)!!!
DNA between genes is repetitive sequences
2 types:
1- short repeated sequences by centromere and at ends of chromosomes by telomeres- protect against DNA loss
2- 2nd type- long repeated sequences
Scattered around genome
“jumping genes”- transposons- can jump into middle of gene and disrupt expression

30. Barbara McClintock Worked at Cold Spring Harbor Lab
Experimented with Indian corn kernel color and transposons

31. Uses in medicine: Therapeutic hormones- insulin, HGH
PCR can help find viruses
DNA microarrays- allows treatment to be tailored to a specific disorder
Vaccines- harmless variant or derivative of a pathogen (usually bacteria or virus)
Stimulates immune system to develop defenses against the pathogen
1 way- produce harmless artificial mutant of pathogen
Causes fewer side effects
2nd way- engineer cells to produce protein that is found on pathogen’s surface

32. Gene Therapy Altering the afflicted individuals genes
New allele can be inserted into somatic cells of infected tissue
Bone marrow cells- good candidate because they include stem cells that can differentiate completely
Bone marrow cells that have been infected with gene carrying virus are injected into afflicted individual
New ideas focus on adding genes to help not fix
Ex: heart growing new blood vessels
There are ethical and technical problems

34. Human Genome Project HGP Goal to sequence the whole human genome
They succeeded
Gene mapping then physical mapping the DNA sequencing