DNA Microarray Technology

Isolate mRNA from tissue sampleNucleic acid molecules hybridize to other nucleic acid molecules that have similar sequences

Make labeled cDNA copy from mRNA template

Cells in the body differentiate due to differential gene expression patterns

A DNA microarray can be used to quantify the relative amount of gene expression in a cell

Each microarray has a complete representation of an organism’s genome

Hybridize labeled cDNA to DNA molecules attached to microarray

Scan DNA microarray

Analyze data Look for fluorescent signals that indicate how many labeled cDNA molecules have bound to the microarray

Stronger signals suggest more of that particular mRNA molecule are expressed in the cell

Do Now - Understanding Isotopes

Your research advisor wants you to design an experiment that you could use to measure the rate of protein synthesis in a cell line that your lab works with routinely.

2. Choose an isotope that you might use to label this biological molecule. Be as specific as you can.

1. What type of biological molecule might you choose to radioactively label?

3. What could you measure to determine the rate of protein synthesis?

Radioactive Isotopes - Pulse/Chase ExperimentSpontaneously give off particles and energy

APPLICATION Scientists use radioactive isotopes to label certain chemical substances, creating tracers that can be used to follow a metabolic process or locate the substance within an organism. In this example, radioactive tracers are being used to determine the effect of temperature on the rate at which cells make copies of their DNA.

DNA (old and new)

Ingredients includingRadioactive tracer (bright blue)

Human cells

Incubators1 2 3

4 5 6

987

10°C 15°C 20°C

25°C 30°C 35°C

40°C 45°C 50°C

TECHNIQUE

2

1

The cells are placed in test tubes, their DNA is isolated, and unused ingredients are removed.

1 2 3 4 5 6 7 8 9

Ingredients for making DNA are added to human cells. One ingredient is labeled with 3H, a radioactive isotope of hydrogen. Nine dishes of cells are incubated at different temperatures. The cells make new DNA, incorporating the radioactive tracer with 3H.

Radioactive Isotopes - Pulse/Chase Experiment

Temperature

The frequency of flashes, which is recorded as counts per minute, is proportional to the amount of the radioactive tracer present, indicating the amount of new DNA. In this experiment, when the counts per minute are plotted against temperature, it is clear that temperature affects the rate of DNA synthesis—the most DNA was made at 35°C.

1 2 3 4 5

Optimumtemperaturefor DNA

30

20

10

0

Cou

nts

per

min

ute

RESULTS

3

RESULTS

A solution called scintillation fluid is added to the test tubes and they are placed in a scintillation counter. As the 3H in the newly made DNA decays, it emits radiation that excites chemicals in the scintillation fluid, causing them to give off light. Flashes of light are recorded by the scintillation counter.

Chapter 2The Chemical Context of Life

AP BiologyParkway Central H.S.A. Bergeron

Hierarchy of Biological Order

Most Common Elements Found in the Human Body

P.C.H.S.N.O.Parkway Central High

School is Number One!

(a) Nitrogen deficiency (b) Iodine deficiency

The Effects of Essential Element Deficiencies

Thyroid Signaling Pathway

Iodine is essential to the synthesis of T3 and T4 (thyroid hormones)

In the absence of iodine, the pituitary gland releases additional TSH

As a result of the increase in TSH, the thyroid gland grows larger to compensate for the reduction in thyroid hormone synthesis

The enlarged thyroid glands often present as a goiter

Atomic number =number of protons

Mass number =Number of protons

+ number of neutrons

Simplified Models of the Atom

Nucleus

(a) (b)In this even more simplifiedmodel, the electrons areshown as two small bluespheres on a circle around thenucleus.

Cloud of negativecharge (2 electrons)

Electrons

This model represents theelectrons as a cloud ofnegative charge, as if we hadtaken many snapshots of the 2electrons over time, with eachdot representing an electron‘sposition at one point in time.

Electron Configurations of First Eighteen Elements

Electron Orbitals

How to Fill Orbitals with Electrons

s orbitals can hold a maximumof 2 electrons

p orbitals can hold a maximum of6 electrons

d orbitals can hold a maximum of10 electrons

f orbitals can hold a maximum of18 electrons

# in front of the orbital refers to the energy level of the orbital

Effects of Electron Excitation

Chemiluminescence - Glow-in-the-Dark Clorox

A ball bouncing down a flightof stairs provides an analogyfor energy levels of electrons,because the ball can only reston each step, not betweensteps.

(a)

Types of Inter- and Intramolecular Bonds found in Biological Molecules

1. Covalent bonds

2. Ionic/Electrostatic bonds

3. Polar covalent bonds -Hydrogen bonds

4. Van der Waals force

Covalent Bonding in Four (4) Molecules

(a)

(b)

Name(molecularformula)

Electron-shell

diagram

Structuralformula

Space-fillingmodel

Hydrogen (H2). Two hydrogen atoms can form a single bond.

Oxygen (O2). Two oxygen atoms share two pairs of electrons to form a double bond.

H H

O O

Single and Double Covalent Bonds

Name(molecularformula)

Electron-shell

diagram

Structuralformula

Space-fillingmodel

(c)

Methane (CH4). Four hydrogen atoms can satisfy the valence ofone carbonatom, formingmethane.

Water (H2O). Two hydrogenatoms and one oxygen atom arejoined by covalent bonds to produce a molecule of water.

(d)

HO

H

H H

H

H

C

Covalent Bonding in Compounds

Biological Importance of Covalent BondingEnergy required to break covalent bonds varies considerably(50-110 kcal/mole) depending on atoms involved and number of bonds that exist between atoms! single bonds < double bonds < triple bonds

Relationship between type of atom and # of covalent bonds

AtomHydrogenOxygenSulfur

NitrogenCarbon

# Covalent bonds12234

Ionic (Electrostatic) Bonding in Sodium Chloride

Formation of Ions

Stable atom + electron (e-) <--> Anion (Negatively charged ion)! Cl + e- --> Cl-

Stable atom - electron (e-) <--> Cation (Positively charged ion)! Ca --> Ca+2 + 2 e-

Biological Importance of Ionic BondingBonds exert attractive force over distance greater than other chemical bonds

Attractive force extends in all directions

Strength of an ionic bond is strong in a non-aqueous environment! 80 kcal/mole required to break ionic bonds in a solid

Charged particles reduce the strength of ionic interactions! -Formation of a “neutralizing coat”! -Formation of a solvent cage/hydration sphere in H2O

A significantly lower amount of energy (1-3 kcal/mole) is required to break ionic bonds in aqueous solution

ElectronegativityThe attraction of a particular kind of atom for the electrons in a covalent bond

In a nonpolar covalent bond–The atoms have similar electronegativities –Share the electron equally

The more electronegative an atom the more strongly it pulls shared electrons toward itself

Polar Covalent Bonding in Water Molecules

Polar covalent bond - Special type of covalent bond that is theresult of unequal electron sharing ! -e- may spend more time near the nucleus of one atom ! than another

Solvent Cages/Hydration Spheres (Water)

• Water molecules overpower the ionic bond above between Na+Cl- by forming hydration spheres.

• Note the orientation of water molecules: negative pole faces the Na+ ion, positive pole faces the Cl-

Hydrogen BondingSimilar to polar covalent bondsbut invariably include hydrogenatoms as part of the molecule

Hydrogen bonds are most commonly found between hydrogen and oxygen, nitrogen,or sulfur atoms

Hydrogen bonds are relativelyweak (2-5 kcal/mole requiredfor breakage)

A collection of hydrogen bonds can create a formidable molecular force

Van der Waals ForceA weak (1 kcal/mole) intermolecular force found between some biological molecules

VDF can be attractive or repulsivedepending on atomic position

Attractive VDF results from momentary, chance inequalities inthe distribution of electrons in a covalent bond! -VDF is different from a polar covalent bond that is due! to differences in the electronegativity of 2+ atoms

VDF becomes stronger as two atoms approach more closely! -Optimum VDF is created at the Van der Waals radius

If atoms are too close to one another, valence e- begin to repeleach other and atoms are pushed apart

• Weak chemical bonds reinforce the shapes of large molecules and help molecules adhere to each other

• The precise shape of a molecule is usually very important to its function in the living cell and is determined by the positions of its atoms’ valence orbitals

• Molecular shape determines how biological molecules recognize and respond to one another with specificity

The Importance of Molecular Shape

Molecular Mimicry

Molecular Mimicry

L-carvone D-carvone

Mint Caraway

Morphine

Carbon

Hydrogen

Nitrogen

Sulfur

OxygenNaturalendorphin

(a) Structures of endorphin and morphine. The boxed portion of the endorphin molecule (left) binds toreceptor molecules on target cells in the brain. The boxed portion of the morphine molecule is a close match.

(b) Binding to endorphin receptors. Endorphin receptors on the surface of a brain cell recognize and can bind to both endorphin and morphine.

Naturalendorphin

Endorphinreceptors

Morphine

Brain cell

The Importance of Molecular Shape

Molecular Shape and Its Biological Implications