Chapter 4 & 5: Nucleic Adics

Question 1

tRNA structure

Answer 1

tRNA has template recognition site called anticodon that recognizes the mRNA codon

Amino acid attaches to the 3’ terminus called the CCA arm via an esther bond

Question 2

Southern Blotting

Answer 2

Used to determine if a specific DNA sequence is present and on which fragment

Question 3

Northern Blotting

Answer 3

A laboratory analysis method used to study RNA

Question 4

DNA Sequencing

Answer 4

Controlled termination of replication is a technique used to sequence DNA

A small amount of 2’, 3’ dideoxy analog is included in a reaction mixture to terminate DNA synthesis

Separation then performed via capillary electrophoresis and visualization via fluorescent tags on the ddNTP

Question 5

Solid-Phase DNA Synthesis

Answer 5

Allows for the synthesis of novel DNA sequences

Question 6

PCR

Answer 6

Can enhance single DNA molecule of interest

Three steps of PCR:

1. Denature – Raise to 95 °C for 15 s to separate DNA
2. Anneal – Lower to ~55 °C to attach primers; usually 20 to 30 nucleotides long
3. Extend – Raise to 72 °C for heat-stable polymerase to work such as Taq polymerase

These steps are repeated 20-40 times in a cycle before cooling down to 4°C

Question 7

Cloning Vectors

Answer 7

Used to get multiple copies of DNA

Composed of various components:

• Origin of replication
• Antibiotic resistance
• Restriction site
• Reporter gene (like LacZ)

Can be:

• Plasmids
• Phages
• Bacterial Artificial Chromosomes (BAC)
• Yeast Artificial Chromosomes (YAC)

Question 8

Insertional Inactivation

Answer 8

Inserted gene disrupts LacZ gene which allows for easy visualization of colonies that express the targeted gene

White = Gene inserted
Blue = No gene inserted

Question 9

Expression Vector

Answer 9

Used to express foreign DNA products i.e., mRNA or proteins

Contain all the components of a normal cloning vector with the addition of:

• Promoter region
• Enhancer region
• Stop and start sites

Can be plasmids or sometimes viruses

Question 10

Next Generation Sequencing

Answer 10

Allows for rapid determination of genomic sequences

Reversible terminator sequencing- similar to Sanger sequencing

Pyrosequencing- detects pyrophosphate (PPi)

Ion semiconductor sequencing- detects protons

Question 11

Genomic Library

Answer 11

A collection of the total genomic DNA from a single organism

DNA is stored in a population of identical vectors, each containing a different insert of DNA

Question 12

cDNA Library

Answer 12

Complementary DNA for all of the mRNA that is contained in a cell

Question 13

Gene Quantification

Answer 13

qPCR- measures amounts of fluorescent copies of DNA

DNA microarray

• Good for lots of samples
• Helpful for cancer research

Question 14

Recombinant DNA

Answer 14

Recombinant DNA can be introduced to animal cells by a variety of means:

1. Foreign DNA precipitated by calcium phosphate can be taken up by animal cells and DNA can become integrated into the chromosome; easy to do but doesn’t work very well
2. DNA can be microinjected into cells: hard to do and maybe 2% effective
3. Viruses can introduce new genes into animal cells – very effective

Question 15

Gene Disruption

Answer 15

Genome editing gives clues to function and new therapies

Knocking out a gene of interest often provides clues for importance

Question 16

CRISPR-Cas System

Answer 16

Can be used for sequence-specific DNA cleavage

CRISPR is a nuclease that can be guided to targeted DNA areas

Works via guide RNA that targets specific sequence of interest