Exam 1 Content

Question 1

What is molecular biology?

Answer 1

The study of gene (and protein) structure and function.

Question 2

Essential toolbox for molecular bio

Answer 2

1. Cell culture
2. PCR & cloning
3. DNA sequencing & BLAST
4. SDS-PAGE & Western Blot (immunostaining)
5. Genome modification

Question 3

Proteins are the _____ of western blot

Answer 3

Workhorse

Question 4

What do the dark bands in a western blot represent?

Answer 4

A lot of protein is present

Question 5

What does it mean when a band in a western blot go from darker to lighter (and vise versa) over time?

Answer 5

darker –> lighter = specific protein expression decreases

lighter –> darker = specific protein expression increases

Question 6

What toolbox does western blot involve?

Answer 6

Cell culture, SDS-PAGE, and western blot

Question 7

Cell culture….

Answer 7

• Source material (human, transformed organism, unmodified organism)
• Allows propagation of biomolecule of interest

Question 8

PCR & Cloning…..

Answer 8

• Making millions of copies of a DNA specific sequence

-Manipulating DNA sequence

Question 9

DNA Sequencing…

Answer 9

• Obtaining the sequence of monomers in a biomolecule
• BLAST: search tool database, used to compare DNA sequences for similarities between organisms

Question 10

SDS-PAGE & Western Blot…

Answer 10

• Allows separation of molecules based on size
• Allows visualization of a specific biomolecule in a mixed population of molecules
• Antibodies used to find specific proteins

Question 11

Immunostaining….

Answer 11

• Using conjugated antibody to visualize a biomolecule of interest
• EXAMPLES (Western Blot, ELISA, Immunohistochemistry, Immunocytochemistry)

Question 12

ELISA…

Answer 12

Testing for molecules in a fluid (urine, blood, spit, etc.)

Question 13

Immunohistochemistry….

Answer 13

Testing for molecules in a tissue

Question 14

Immunocytochemistry….

Answer 14

• Testing for molecules in a cell, and allows us to see whether they are in cytoplasm or nucleus
• Toolbox: DNA manipulation, cloning, cell culture, immunocytochemistry

Question 15

Stem cells have _____ that allow them to not differentiate into different cells

Answer 15

Masters regulators

Question 16

Example: Western Blot –> Irreversible Post-Translational Modifications

Answer 16

• Proteins that get cleaved
• Tested by comparing band intensity of protein before its cleaved to band intensities of protein subunits after cleaved

Question 17

DNA synthesis in vitro

Answer 17

Arthur Kornberg

Question 18

RNA synthesis in vitro

Answer 18

Jerard Hurwitz

Question 19

Protein Synthesis in vitro

Answer 19

Paul Zemecnik

Question 20

Marshall Nirenberg

Answer 20

Cracked the genetic code

Question 21

DNA composition and structure (people)

Answer 21

Chargaff, Franklin, Watson, Crick

Question 22

Transformation (people who tested for it)

Answer 22

Griffith, Avery, Hershey and Chase

Question 23

DNA Replication (proved mechanism)

Answer 23

Meselson and Stahl

Question 24

PCR and DNA sequencing (people)

Answer 24

Mullis and Sanger

Question 25

Transforming Principle

Answer 25

A substance that can be transferred from nonliving cells to living cells, causing living cells to show characteristics of the nonliving cell.

Question 26

Frederick Griffith experiment (explain)

Answer 26

transformed a harmless strain of streptococcus (rough strain) into a pathogenic strain (smooth), *but he didn't show which biological molecule was responsible.* 1.) Rough strain injected into mouse = mouse lives (rough strain recovered) 2.) Smooth strain injected into mouse = mouse died (smooth strain recovered) 3.) Smooth strain cell-free extract + rough strain injected into mouse = mouse died (smooth and rough strain recovered)

Question 27

Oswald Avery Experiment (explain)

Answer 27

Identified (*indirectly*) the "transforming principle" is a nucleic acid using enzymes 1.) Smooth strain cell-free extract + rough strain injected into mouse + DNAse = only rough strain recovered 2.) Smooth strain cell-free extract + rough strain injected into mouse + w/o DNAse = both rough and smooth strain recovered

Question 28

Stable Isotopes

Answer 28

Distinguished/identified only by a difference in mass

Question 29

Unstable isotopes

Answer 29

Identified by measuring radioactivity/energy that they release

Question 30

Centrifuge

Answer 30

Instrument that rotates samples at high velocities to separate components by denisty Pellet = high density Supernatant = lower density

Question 31

Alfred Hershey and Marsha Chase experiment (explain)

Answer 31

Identified (directly) the transforming principle as a nucleic acid using unstable isotopes and bacteriophages 1.) Generate two batches of radioactive phage. One with radioactive methionine (protein coat of bacteriophage) and the other with radioactive bacteriophage DNA 2.) Binding reaction (radioactive phage + bacterial cells) 3.) Knocked virus off bacteria used a blender 4.) Identify which isotope entered bacteria using a centrifuge - Phage grown with radioactive protein coat = radioactivity in supernatant (not in cell) - Phage grown with radioactive DNA = radioactivity in pellet (inside cell)

Question 32

Matthew Meselson and Franklin Stahl experiment (explain)

Answer 32

Showed that DNA replication was semiconservative using stable isotopes 1.) Grow bacteria in media containing N15 (heavy isotope) = lots of DNA containing N15 after replication 2.) Shift bacteria to media containing N14 (light isotope) = DNA shifting from containing N15 only to containing both N15 & N14, then after time containing majority N14 and little N15

Question 33

What are cell-free extracts?

Answer 33

Content inside a cell that is needed to replicate DNA or express a gene

Question 34

Kary Mullis experiment (explain)

Answer 34

Used a thermostable DNA polymerase to make PCR more efficient High temps are required to denature DNA strands. A thermostable bacteria contains a DNA polymerase that is not denatured at high temps and can function at high temps.

Question 35

Modified Nucleotides

Answer 35

*Chain terminating* nucleotides that lack the 3'-OH group that is required for the formation of the phosphodiester bond between two nucleotides. This causes DNA polymerase to cease synthesis of the new DNA strand. Fluorescently or radioactively labeled for detection

Question 36

Fredrick Sanger experiment (explain)

Answer 36

Used chain terminating nucleotides to read the sequence of a DNA molecule 1.) 4 separate sequencing rxn's, each w/ one of 4 fluorescently conjugated chain terminating nucleotides 2.) 4 groups of fragments that end in 1 of 4 colors (bases). "Read" the DNA sequence from the bottom of the gel to the top of the gel.

Question 37

DNA Replication vs. PCR

Answer 37

DNA replication: - in vivo - Duplicate entire genome (2x) - involve enzymes & accessory proteins - *use helicase* for DNA strand separation PCR - in vitro - amplify a specific DNA sequence (2x) - involves some enzymes - *use high heat* for DNA strand separation