Analytical techniques

Question 1

Name 4 ways of investigating/sequencing DNA sequences or libraries.

Answer 1

1. Controlled termination method of replication (sanger sequencing)
   
   1. Sequencing: Stop addition of new nucleotide base after each one.
2. Screening DNA library with probes
3. Next generation sequencing methods
   
   1. Pyrosequencing
   2. Ion semiconductor sequencing
   3. Reversible termination method
4. DNA microarray
   
   1. Visually see what cDNA stuff attaches to

Question 2

Name 3 methods of DNA synthesis

Answer 2

1. Automated solid phase methods
   
   1. A DNA strand synthesis by adding one base at a time
2. PCR
   
   1. amplification and replication of a DNA sequence of interest
3. Reverse transcriptase
   
   1. make cDNA from mRNA

Question 3

Name 14 DNA editing tools

Answer 3

1. Restriction enzymes
   
   1. Cut DNA at restriction sites, form sticky ends
2. Bacterial Plasmid vector
   
   1. Plasmid inserts DNA
   2. cloning vector
   3. Expression vector
3. λ-Phage vector
   
   1. Bacterial virus inserts DNA into cell
   2. Lytic pathway
   3. Lysogenic pathway
4. Bacterial artificial chromosome (BAC)
   
   1. Engineered E.Coli F factor, for large DNA insertions
5. Yeast artificial chromosome (YAC)
   
   1. For even larger inserts
6. Site directed mutagenesis
   
   1. Replace a single nucleotide base
7. Cassette mutagenesis
   
   1. Remove section of plasmid, insert custom section
8. Mutagenesis by reverse PCR
   
   1. Can do a reverse PCR, removing a segment rather than amplifying it.
9. Designer genes
   
   1. Splicing together genes that would never be expressed together normally
10. Retrovirus
    
    1. Sort of a combination of lysogenic and lytic pathway viruses
11. Gene knockout
    
    1. Make it so that an organism doesn’t express a gene
12. Genome editing
    
    1. Engineered endonucleases cause specific dsDNA breaks
13. RNA interference
    
    1. Genes in normal genome will not be expressed if they match genes in inserted dsRNA
14. Ti plasmids
    
    1. Can be used to insert genes in plants.

Question 4

Define Salting out

Answer 4

1. Separates based on solubility.
2. Most proteins are less soluble at high salt concentrations.
3. Because the salt concentration differs from protein to protein, this can be used to differentially separate them.
4. Can be combined with dialysis to remove the salt.

Question 5

Define differential centrifugation

Answer 5

1. Releases proteins from cell to be purified
2. Homogenate formed by disrupting cell membrane
3. cenrrifugation is used to differentiate pellet, with denser material going to the bottom first.

Question 6

Define Dialysis

Answer 6

1. Seperates proteins from small molecules
2. Relies on concentration gradient and a semi-permeable membrane.
3. Useful for removing salt and other small molecules, but will not differentiate proteins.

Question 7

Draw diagram of both indirect and direct ELISA

Answer 7

Question 8

Describe or draw the solid phase method of polypeptide synthesis.

Answer 8

Question 13

Describe the basis of X-Ray Crystallography, its resolution, what is needed to do it.

Answer 13

1. Basis
   
   1. Crystallize protein by salting out
   2. Shoot with X-rays
   3. Electrons scatter X-rays
   4. Scattered waves recombine
   5. The way they recombine depends on atomic arrangement.
   6. A fourier transform equation allows visualization on an electron density map.
2. Resolution - Atomic to near atomic
3. Needs:
   
   1. High concentration of pure protein
   2. Non-membrane protein
   3. Protein needs to be able to crystallize
   4. It is kind of hard to do well.

Question 14

Describe the basis of NMR Spectroscopy, its resolution, what is needed to do it.

Answer 14

1. Basis
   
   1. Transition between spin states of atoms affected by electromagnetic radiation.
   2. Different frequencies of EM radiation, called chemical shifts help determine the atomic composition of a protein.
   3. Different atoms react to EM radiation differently.
2. Resolution - near atomic
3. Needs:
   
   1. Protein can be in solution
   2. need a lot of protein
   3. can do membrane proteins
   4. better with smaller proteins.

Question 15

Name 12 separation techniques.

Answer 15

1. Homogenization and centrifugation
   
   1. Releases proteins from cell and separates based on density
2. Salting out
   
   1. Based on salt solubility
3. Dialysis
   
   1. Semi-permeable membrane and concentration gradient
4. Gel-filtration chromatography
   
   1. Size of particles
5. Ion-exchange chromatographyCation exchange
   
   1. Attracts positive charged proteins
6. Anion exchange
   
   1. Attracts negatively charged proteins
7. Affinity chromatography
   
   1. Matrix has affinity for specific groups
8. HPLC
   
   1. High pressure, high resolution chromatography.
9. Gel Electrophoresis
   
   1. Moves proteins based on molecular weight
10. Isoelectric focusing
    
    1. Separates based on pI
11. 2D Electrophoresis
    
    1. Separates based on both pI and weight
12. Ultracentrifugation
    
    1. Separates based on sedimentation coefficient

Question 16

Name 8 immunology based protein analysis techniques.

Answer 16

1. Antibody (immunoglobulin) preparation
   
   1. Antigen injected into animal. Serum taken and antibody purified.
2. Monoclonal antibodies
   
   1. Identical. Recognize one specific epitope on antigen
3. Polyclonal antibodies
   
   1. Different. Recognize different epitopes on an antigen.
4. ELISA
   
   1. Enzyme that produces color in presence of substrate. linked to antibody for antigen. Presence of antigen means that enzyme will break substrate, producing color.
5. Indirect ELISA
   
   1. Antigen coated well. Antibody 1 links to antigen. Antibody 2 with enzyme links to antibody 1.
6. Sandwich ELISA
   
   1. Antibody 1 coated well. Antigen links to antibody 1. Antibody 2 with enzyme links to antigen.
7. Western blotting.
   
   1. Electrophoresis. Transfer to membrane. Stain with fluorescent linked antibody for protein of interest. Uses 1st and 2ndary antibody.
8. Fluorescence microscopy
   
   1. Fluorescent markers attach to specific things in cell. Look at with microscope.

Question 17

How does the CRISPR/Cas9 system work and what is it used for.

Answer 17

1. It is used in genome editing

Question 18

Name 9 protein sequencing techniques.

Answer 18

1. Mass spectrometry
   
   1. Uses mass/charge ratio to determine identity of molecule
2. MALDI (matrix assisted laser desorption/ionization)
   
   1. Charges molecules
3. ESI (Electrospray ionization)
   
   1. Charges molecules
4. TOF (Time of flight)
   
   1. Analyzes mass by measuring time to get across a chamber
5. Edman degradation
   
   1. Determines amino acid sequence by labeling and removing 1 amino acid at a time. Mass and identity determined by mass spec.
6. Tandem Mass spectrometry
   
   1. Multiple mass spec lined up in a row to analyze chains then smaller particles.
7. Proteolytic cleavage
   
   1. Chemical and enzymatic cleavage at specific points allows determination of sequence
8. Overlap peptides
   
   1. Sequence can be determined by cleaving with different things and seeing where fragments overlap.
9. Genomic methods
   
   1. Use known genetic info to determine polypeptide sequence

Question 19

Name 3 Protein 3D structure analytical techniques.

Answer 19

1. X-ray Crystallography
   
   1. How the electrons scatter x-rays shows structure.
   2. Sample must be crystallized
   3. Need highly concentrated sample
   4. Need pure sample
   5. Doesn’t work well on membrane proteins.
2. NMR Spectroscopy
   
   1. How a magnetic field affects spin of atoms shows structure
   2. Can be used on native, in solution
   3. Need lots of sample
3. Cryo-EM
   
   1. Super cold, allows imaging of native form non stained.

Question 22

Describe the basis of Cryo EM, its resolution, what is needed to do it.

Answer 22

1. Basis
   
   1. Is a form of transmission electron microscopy where the molecules are frozen at very low temperatures.
2. Resolution - near atomic
3. Needs:
   
   1. Cold
   2. can be at native, unstained.

Question 23

Describe co-immunoprecipitation, what is it used for?

Answer 23

1. Used to investigate protein-protein interactions
2. Pull down an entire complex using an antibody for a known part
3. then can investigate if any other known proteins are attached with other antibodies or other methods

Question 24

Draw diagram of both indirect and direct ELISA

Answer 24

Question 25

Describe or draw the solid phase method of polypeptide synthesis.

Answer 25

Question 29

Name 12 separation techniques.

Answer 29

1. Homogenization and centrifugation
   
   1. Releases proteins from cell and separates based on density
2. Salting out
   
   1. Based on salt solubility
3. Dialysis
   
   1. Semi-permeable membrane and concentration gradient
4. Gel-filtration chromatography
   
   1. Size of particles
5. Ion-exchange chromatographyCation exchange
   
   1. Attracts positive charged proteins
6. Anion exchange
   
   1. Attracts negatively charged proteins
7. Affinity chromatography
   
   1. Matrix has affinity for specific groups
8. HPLC
   
   1. High pressure, high resolution chromatography.
9. Gel Electrophoresis
   
   1. Moves proteins based on molecular weight
10. Isoelectric focusing
    
    1. Separates based on pI
11. 2D Electrophoresis
    
    1. Separates based on both pI and weight
12. Ultracentrifugation
    
    1. Separates based on sedimentation coefficient

Question 30

Describe the basis of X-Ray Crystallography, its resolution, what is needed to do it.

Answer 30

1. Basis
   
   1. Crystallize protein by salting out
   2. Shoot with X-rays
   3. Electrons scatter X-rays
   4. Scattered waves recombine
   5. The way they recombine depends on atomic arrangement.
   6. A fourier transform equation allows visualization on an electron density map.
2. Resolution - Atomic to near atomic
3. Needs:
   
   1. High concentration of pure protein
   2. Non-membrane protein
   3. Protein needs to be able to crystallize
   4. It is kind of hard to do well.

Question 31

Describe the basis of NMR Spectroscopy, its resolution, what is needed to do it.

Answer 31

1. Basis
   
   1. Transition between spin states of atoms affected by electromagnetic radiation.
   2. Different frequencies of EM radiation, called chemical shifts help determine the atomic composition of a protein.
   3. Different atoms react to EM radiation differently.
2. Resolution - near atomic
3. Needs:
   
   1. Protein can be in solution
   2. need a lot of protein
   3. can do membrane proteins
   4. better with smaller proteins.

Question 32

Name 8 immunology based protein analysis techniques.

Answer 32

1. Antibody (immunoglobulin) preparation
   
   1. Antigen injected into animal. Serum taken and antibody purified.
2. Monoclonal antibodies
   
   1. Identical. Recognize one specific epitope on antigen
3. Polyclonal antibodies
   
   1. Different. Recognize different epitopes on an antigen.
4. ELISA
   
   1. Enzyme that produces color in presence of substrate. linked to antibody for antigen. Presence of antigen means that enzyme will break substrate, producing color.
5. Indirect ELISA
   
   1. Antigen coated well. Antibody 1 links to antigen. Antibody 2 with enzyme links to antibody 1.
6. Sandwich ELISA
   
   1. Antibody 1 coated well. Antigen links to antibody 1. Antibody 2 with enzyme links to antigen.
7. Western blotting.
   
   1. Electrophoresis. Transfer to membrane. Stain with fluorescent linked antibody for protein of interest. Uses 1st and 2ndary antibody.
8. Fluorescence microscopy
   
   1. Fluorescent markers attach to specific things in cell. Look at with microscope.

Question 33

Name 9 protein sequencing techniques.

Answer 33

1. Mass spectrometry
   
   1. Uses mass/charge ratio to determine identity of molecule
2. MALDI (matrix assisted laser desorption/ionization)
   
   1. Charges molecules
3. ESI (Electrospray ionization)
   
   1. Charges molecules
4. TOF (Time of flight)
   
   1. Analyzes mass by measuring time to get across a chamber
5. Edman degradation
   
   1. Determines amino acid sequence by labeling and removing 1 amino acid at a time. Mass and identity determined by mass spec.
6. Tandem Mass spectrometry
   
   1. Multiple mass spec lined up in a row to analyze chains then smaller particles.
7. Proteolytic cleavage
   
   1. Chemical and enzymatic cleavage at specific points allows determination of sequence
8. Overlap peptides
   
   1. Sequence can be determined by cleaving with different things and seeing where fragments overlap.
9. Genomic methods
   
   1. Use known genetic info to determine polypeptide sequence

Question 34

How does the CRISPR/Cas9 system work and what is it used for.

Answer 34

1. It is used in genome editing

Question 35

Name 3 Protein 3D structure analytical techniques.

Answer 35

1. X-ray Crystallography 1. How the electrons scatter x-rays shows structure. 2. Sample must be crystallized 3. Need highly concentrated sample 4. Need pure sample 5. Doesn't work well on membrane proteins. 2. NMR Spectroscopy 1. How a magnetic field affects spin of atoms shows structure 2. Can be used on native, in solution 3. Need lots of sample 3. Cryo-EM 1. Super cold, allows imaging of native form non stained.

Question 38

Describe the basis of Cryo EM, its resolution, what is needed to do it.

Answer 38

1. Basis 1. Is a form of transmission electron microscopy where the molecules are frozen at very low temperatures. 2. Resolution - near atomic 3. Needs: 1. Cold 2. can be at native, unstained.