Biotech and Microscopy

Question 1

What are the two broad categories of microscopy?

Answer 1

Optical & Electron

Question 2

What are the two types of optical microscopy?

Answer 2

Compound and fluorescence

Compound involves killing cells and staining.
Flurescence involves using a fuorescent marker to tag structures and can be used on living samples.

Question 3

What are the types of electron microscopy?

Answer 3

Scanning (SEM) and Transmission (TEM)
Scanning: produces 3D image and ideal for viewing external surfaces of cells/tissues/molecules also involves killing cells by dehydration and coating.
Transmission: high magnification 2D image and ideal for viewing internal structures of cells/tissues/organelles, also involves killing sample by dehydrating and fixing.

Pro tip -> SEM -> Think S for Surface

Question 4

What is cell fractionation and its process?

Answer 4

Uses centrifucation to separate a cell’s organelles based on density and size from largest to smallest.
Process:
1. Homogenization - break cells apart
2. Low-speed centrifugation - nuclei separates
3. Medium-speed centrifugation - mitochondria/chloroplasts separates
4. High-speed centrifugation - microsomes & viruses separate.

Question 5

What are the three main types of horizontal gene transfer?

Answer 5

Conjugation (pilus), Transduction (virus), Transformation (absorption)

Question 6

What is the process of recombining DNA

Answer 6

1. Use of restriction enzyme at palindromic sequences (sticky ends)
2. Combine DNA with Ligase

Question 7

What is restriction fragment polymorphisms (RFLPs)

Answer 7

Location of restriction sites on human DNA varies between individuals which will create unique fragments of different sizes when applying restriction enzymes.

Question 8

What are single nucleotide polymorphisms (SNPs)

Answer 8

Single nucleotide differences in the human genome

Question 9

What is the purpose of reverse trancription?

Answer 9

To produce complimentary DNA (cDNA) out of mature mRNA that does not have any introns. cDNA is more stable than mRNA.

Used by retroviruses like HIV and Hep B.
Used to make insulin.

Question 10

What are the steps of PCR?

Answer 10

1. Denaturation (94)
2. Annealing (45-54) allows primers to attach
3. Elongation (65) w/ prokaryotic polymerase

Question 11

How does DNA microarray assay work?

Answer 11

Compares DNA from different cells (normal vs cancerous)
Extract mRNA and transcribe cDNA. Place cDNA in wells that are loaded with DNA sequences of certain genes. Can evaluate to see what genes are turned on in cancer cells vs normal cells. The cDNA is tagged with fluorescent markers.

Question 12

What are the steps for blotting?

Answer 12

1. Extraction
2. Separation with electrophoresis
3. Transferring to nitrocellulose paper
4. Expose paper to labelled probe
5. Visualization

SNOW DROP

Question 13

What are the three steps to immunofluorescent staining?

Answer 13

1. Addition of primary antibody
2. Addition of secondary antibody
3. Visualization of protein of interest

Question 14

What is in-vivo mutagenesis?

Answer 14

Helps determine the function of a gene by introducing mutation into a gene and observe phenotypic differences.

Question 15

What is genome annotation?

Answer 15

Matching up unknown DNA sequences to known sequences to evaluate what the gene function is.

Question 16

How is a genomic library formed?

Answer 16

1. Isolate gene of interest
2. Cut genome with restriction enzymes
3. Cut plasmid with same restriction enzymes
4. Ligate genes into plasmid
5. Insert plasmid into bacteria (via transformation) w/ electroporation or heat shock
6. Allow bacteria to multiply
7. Isolate DNA as needed.

Question 17

What are the steps to mammalian reproductive cloning?

Answer 17

1. Isolate donor cell with nucleus
2. Isolate unfertilized enucleated egg
3. Transplant nucleus into enucleated egg
4. Embryo formation
5. Transfer embryo into surrogate mother
6. Deliver baby clone

Question 18

What is Pasteur’s swan neck flask experiment?

Answer 18

Experment establishing that life cannot arise spontaneously.

Question 19

What is Griffith’s experiment?

Answer 19

Used two strains of bacteria that were injected into mice: the rough and smooth strain.
The rough strain (r strain) did not have a protective capsule and were non-virulent: they were killed by the mouse’s immune system.
The smooth strain (s strain) had a protective capsule and were virulent: they killed the mouse.
The s strain was then heat-killed and injected: the mouse lived.
The s strain that was heat-killed and added to a solution with the r strain and then injected: the mouse died because the r strain was transformed and produced a protective capsule.

Question 20

What was the Aver-MacLeod-McCarthy expeiment?

Answer 20

Follow up to the Griffith experiment.
Found that DNA was responsible for transforming cells after removing proteins, RNA, and DNA in different samples.

Question 21

Hershey & Chase Experiment?

Answer 21

Bacteriophages were injected with phosphorus labelled DNA or sulfur labelled protein capsules. It was confirmed that DNA was the genetic material used by viruses.

Question 22

What is Meselson & Stahl’s experiment?

Answer 22

Proved the semiconservative replication model was the valid DNA model.
1. Grew E. Coli in a medium with nucleotides
2. Bacteria transferred to medium with 14N
3. 15N Bacteria replicated in new medium
4. Replication continue for another round.

Question 23

What is Gurdon’s Nuclear Transfer Experiment?

Answer 23

Proved that fully differentiated cells do not lose their genetic information - they retain the full genome. He placed a nucleus from a differentiated frog cell into an enucleated egg cell -> gave rise to a new frog.