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KIDDER APPROACH: the discipline dealing with what is good and bad and with moral duty and obligation
End-based: Good for the greatest number
Care-based: Golden Rule "treat others the way you want to be treated
Rule-based: Live by moral or religious code or law

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1. TECHNIQUE LABS
-Micro: very small (.000001)
-Milli: one thousandth (1.0)
-Micropipet: A very fine pipette for measuring, transferring, or injecting, very small quantities of liquid
-Reaction tube: a collapsible cylindrical container of soft metal or plastic closed with a cap, used to hold viscous liquids or pastes.
-microcentrifuge: Used to rapidly spin and separate SMALL samples of substances of greater and lesser densities
-Electrophoresis: A process where DNA fragments are separated according to size using electrical charges
-Gel Box: Instrument used in electrophoresis and DNA fingerprinting, has gel plates and wells
-Buffer: a solution that minimizes changes in pH when extraneous acids or bases are added to the solution
-Current: A movement of electrical charges around a closed path or circuit
-Agar/Agarose: Gel that separates the different sized fragments of DNA
-Wells: holes in gels were liquid is stored
-Lanes: DNA molecules travel through gel path to get to positive poles
COMPLETE METRIC CONVERSIONS
K [1000]
h[100.]
da[10.]
(m,l,g) [1.0]
d [0.1]
c[.01]
m[.001]
microliter [.000001]
MICROPIPETING
-Using a Micropipette appropriately
1. FIRO and ISRO
2. Do not exceed range (never go beyond or below pipette range)
3. Hold pipette vertically
4. never pipette without tip
5. Release slowly
-Ranges
P-20 (0.5-20 microliters)
P200 (20-200 microliters)
P1000 (200-1000 microliters)
do not exceed or go below range
F- First Stop
I- Insert

R- Release
O-  Out

I-Insert
S- Second stop
R- Release
O- Out
2. PCR
-In vivo: within the living body
-primer: an already existing RNA chain bound to template DNA
-TAQ polymerase: DNA synthesis enzyme that can withstand the high temperatures of PCR
-Buffer: A solution that minimizes changes in pH level when extraneous acids or bases are added to the solution
-dNTPs: deoxynucleotide triphosphates----> it catalyzes a reaction that attaches the nucleotide to the 2nd DNA strand
-Denature 95 C: High temperatures cause hydrogen bonds to break between bases and the two strands separate
-Annealing 55-65 C: Allows primers to attach to specific singled stranded DNA templates complementary in sequence to short sections of DNA on each end of the sequence to be copied.
-Extension 72 C: Heat is increased to enable the new DNA to be made by (Taq) Polymerase in a short amount of time which results in a huge number of copies of DNA

ALU

- Alu is a transposable DNA sequence that "reproduces" by copying itself and inserting into new chromosome locations. (JUMPING GENE)
What can we use Alu insertions to determine?
-compare genotypes between different people through electrophoresis (agarose gel)

Wolbachia

What is Wolbachia Pipientis? Where can you find it?
-Bacteria in insects found in insect cells

How can we determine if an organism is infected with Wolbachia? (what do we use to find it?)
Wolbachia DNA extraction and PCR
chelex, supernatant, bug alcohol, pellet, pellet mix
What is the benefit of Wolbachia for an organism? What is the benefit for humans?
-Gets rid of mosquitos with disease
because Wolbachia is in the cytoplasm
more females than males the more Wolbachia will continue to spread

mtDNA

-structures within cells that convert the energy from food into a form that cells can use
-Inherited from the Mother
what can we use mtDNA to determine?
Crime investigations and provide ancestry information
3. DNA SEQUENCING
Genome
DNA sequencing
DNA ladder
Deoxynucleotide (dNTP)
Dideoxynucleotide (ddNTP)
Chain Terminators: molecules that closely resemble normal nucleotides but lack the essential 3' OH
Single Nucleotide Polymorphism (SNP): represents a difference in a single DNA building block nucleotides (How many nucleotides are different? ONLY ONE!)
Copy Number Variant (CNV) or (CNP copy number Polymorphism) : Number of Copies from one individual to another
4. RESTRICTION DIGEST
1. Cut DNA for cloning
2. Cut DNA to create RFLP's (restriction fragment length polymorphism, used in DNA fingerprinting)
plasmid: A circular piece of DNA derived from bacteria that can be used for recombinant DNA technology
Recombinant DNA- transferring a gene from one organism into another

3 Features

-origin of Replication: Allows plasmid to be copied on its own
-Selectable Marker: selects for bacteria that have the plasmid (usually antibiotic resistance)
-cloning site: an area of the plasmid that DNA can be added to transform a bacteria

Used in DNA fingerprinting

Restriction Enzyme

-Sticky ends: latch on to site, cause shift and breaks away fragments
-Ligation: Ligase "glue" fragments together
-Blunt ends: don't leave an overhang

What is needed to do a restriction digest?

-DNA
-Restriction Enzyme
-Buffer

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1. MOLECULAR STRUCTURE
-Base pairing: Complementary molecules bind only to each other
-Phosphodiester (bonds): the type of bond that links the backbone of DNA together (it is strong)
-Deoxyinbase: the sugar that DNA contains
-sugar-phosphate backbone: A molecule that joins the bases together in DNA using a phosphate bond
-Double helix: the overall structure of DNA
-Anti-parallel: term to describe the orientation of the two strands
-Nuclease: An enzyme that breaks apart DNA
-5 carbon sugar: A molecule that has a ring structure belongs to a carbohydrate
-Nitrogen bases: A molecule that hydrogen bonds with its complementary molecule.
-DNA: A nucleic acid that contains a deonxysbride sugar
-Nucleotide: A molecule made up of three distinct pairs (base, phosphate group, sugar)
-Purine: base with DOUBLE ring structure
-pyrimidine: base with SINGLE ring structure
-cytosine: SINGLE ring, binds with guanine
-guanine: DOUBLE ring, binds with cytosine
-base sequence: The order of the bases within a DNA strand
-Thymine: SINGLE ring, binds with Adenine
-Adenine: DOUBLE ring, binds with Thymine
-RNA: A nucleic acid that contains a ribose sugar
2. DNA EXTRACTION
-EDTA
Role: Weakens membrane
How it works: Attacks proteins in membrane
-SDS (detergent)
Role: Finish membrane breakdown
How it works: Attacking/breaking down lipids and membrane
-Saltwater
Role: Releases DNA from histones
How it works: The sodium attracts DNA more than Histone (separates DNA from histones)
-Ethanol (cold water)
Role: Precipitate DNA
How it works: DNA is not solubles in alcohol
3.DNA REPLICATION
-In vivo: within the living body
-leading strand: DNA synthesis moves CONTINUOUSLY 5'----3' direction
-lagging strand: DNA synthesis move DISCOUNTINOUSLY cannot move in 5'----3' direction so goes backward in short sections
-Okazaki fragments: chunks of replicated DNA
-Replication Fork: A Y-shaped region on a replicating DNA molecule where new strands are growing
-Single stranded binding proteins (SSB): Prevents re-annealing of the DNA strands
-Helicase: unwinds the DNA helix
-DNA polymerase III: Synthesis DNA in a 5'----3' direction (extends RNA in Okazaki fragments)
-Primase: Synthesis a short RNA primer
-RNA primer: Short segments of RNA used to initiate synthesis of a new strand of DNA during replication
-DNA polymerase I (Exonuclease): carries the individual nucleotide to the site of replication

Enzymes involved

-DNA Ligase- Re-anneals the semi- conservative strands and glues the Okazaki fragments on the lagging strand.  (seals sugar phosphate backbone)
-Primase-  Provides a starting point which puts down an RNA primer (polymerase 3 starts to add to the strands)

During separation, the two strands of the DNA double helix uncoil at a specific location called the origin. Several enzymes and proteins then work together to prepare, or prime, the strands for duplication. Finally, a special enzyme called DNA polymerase organizes the assembly of the new DNA strands.