Misc Topics

Question 1

What are restriction enzymes?

Answer 1

• Enzymes used to cut DNA at specific sequences, usually palindromic
• Naturally synthesized by bacteria for an immune response against invading viral bacteriophages
  – The enzymes destroy the phage’s DNA before it can insert into the bacterial chromosome!

Question 2

Are bacteria sensitive to their own restriction enzymes?

Answer 2

No, since the sequences their enzymes recognize and cut are silenced via methylation.

Question 3

What is a palindromic sequence?

Answer 3

A DNA sequence which in 5’-3’ direction reads the same as its complimentary strand read 5’-3’

Question 4

What are northern, southern, & western blots?

Answer 4

SNOW DROP

• Southern → DNA
• Northern → RNA
• Western → Proteins

Question 5

What charge do RNA and DNA carry?

Answer 5

-ve charges

Question 6

What are vectors?

Answer 6

Vectors are carriers of DNA

Question 7

What is a plasmid vector?

Answer 7

Plasmid → circular DNA molecule capable of autonomous replication.

Usually associated with antibiotic resistance!

Question 8

What are phage vectors?

Answer 8

VIruses that infect bacteria. Carry a much longer sequence of DNA than plasmids, so preferred for this reason.

Bacteriophage λ has been used as a phage for a long time.

Question 9

What are genomic libraries?

Answer 9

All DNA (fragmented) from a single host is cloned into a phage.

Question 10

What is a cDNA library?

Answer 10

All mRNA from a specific host cell is made into complementary DNA (cDNA).

This collection is known as the cDNA library.

The cDNA from a brain cell vs. a liver cell will be very different due to different gene transcriptions!

Question 11

What is an expression library?

Answer 11

cDNA from a particular cell type in a host is put on a promoter to make proteins.

This is used to make recombinant proteins in large amounts, eg insulin.

Question 12

What is transfection?

Answer 12

The process whereby you introduce a DNA sequence into a mammalian cell using non-viral methods. Can be done in many different ways:

1. Microinjection
2. Calcium phosphate precipitation
3. Electroporation
4. Cationic lipids
5. Other non-viral methods

Question 13

What are the steps in the DNA polymerase chain reaction?

Answer 13

1. Denaturation: 90C, 10s
2. Annealing: 50-65C, 10s
3. Extension: 70C, 30s

Question 14

Which enzyme catalyzes the PCR process?

Answer 14

Taq polymerase (enzyme resistant to high temps).

Question 15

What is real-time PCR?

Answer 15

Uses fluorescent probes to
Measures the amount of mRNA

Question 16

What is restriction fragment length polymorphism (RFLP)?

Answer 16

Differences among people in their DNA sequences at sites recognized by restriction enzymes. Such variation results in different-sized DNA fragments produced by digesting the DNA with a restriction enzyme. RFLPs can be used as genetic markers, which are often used to follow the inheritance of DNA through families.

Question 17

What is siRNA technology?

Answer 17

It is a double-stranded RNA tech that can selectively silence particular genes.

Question 18

What is shotgun sequencing?

Answer 18

A computer sequences

Question 19

Mouse strains have different susceptibilities to radiation-induced lung cancer. Which strain has a low susceptibility?

Answer 19

C₃H / H₀J

Question 20

How do mutations differ b/w high and low LET radiation?

Answer 20

High LET → Large Mutations
Low LET → Small Mutations

However, looking at a mutation, you cannot tell whether it came from high or low LET RT.

Question 21

What is the Chromatin Immunoprecipitation Assay (ChIP) assay used for?

Answer 21

To detect proteins bound to promoter sequences

Question 22

What are reporter gene assays?

Answer 22

These are genes that researchers attach to a regulatory sequence of another gene of interest..

The reporters are attached to a marker, such as a fluorescent dye, that can identify where the gene promoter is.

Question 23

What’s the use of propidium iodide?

Answer 23

• DNA-intercalating dye
• Stains cell proportionally to the amount of cellular DNA

Question 24

What does the TUNEL assay measure?

Answer 24

Apoptosis

Question 25

What is transduction?

Answer 25

Introduction of nucleic acids or foreign DNA into a cell using viral vectors

Question 26

What’s electroporation?

Answer 26

Introduction of DNA into a cell by using pulsed, high-voltage electricity that can open pores within the cell membrane

Question 27

What is transformation?

Answer 27

The process by which normal cells are transformed into malignant cells

Question 28

What’s Sanger sequencing?

Answer 28

• A DNA replication method, in which the nucleotides that are added to the DNA are observed (the DNA sequence can then be deduced)
• Uses dideoxynucleotide triphosphates (ddATP, ddTTP, ddCTP, ddGTP)

Question 29

What’s conjugation?

Answer 29

The process where a bacteria transfers a plasmid DNA into another baceterai

Question 30

What’s conjugation?

Answer 30

The process where a bacteria transfers a plasmid DNA into another bacteria

Question 31

What is complementation?

Answer 31

Restoring a normal copy of a gene in the setting of biallelic mutations

Question 32

What’s the MISO PET used for?

Answer 32

Detect tissue hypoxia

Question 33

What’s the main function of cos sites?

Answer 33

They are essential for packaging DNA in phages

Question 34

Which enzyme does the clustered regularly interspersed short palindromic repeats (CRISPR) technique?

Answer 34

Cas9