Biotechnology

Question 1

Restriction Endonucleases

Answer 1

Enzymes that break phosphodiester bonds within DNA molecules
Type I: Specific recognition site with nonspecific cleavage site a distance away
Type II: Specific recognition site with internal site cleavage
Type III: Specific recognition site with nearby nonspecific cleavage site

Question 2

Probes

Answer 2

• Complement of the sequence for DNA/RNA
• DNA probes are more stable and preferred
• Target sequence must be single stranded
• Increasing length increases specificity and decreases efficiency
• Visualized through radioactive exposure on a film (P35 or C14) or biotinylated with a fluorescent dye

Question 3

Blotting

Answer 3

Makes a stable copy of a gel transferring nucleic acid or protein to a nitrocellulose membrane
Southern: Used to separate DNA, usually based on length
Northern: Used to separate RNA, usually based on length
Western: Used to separate proteins based on various attributes and visualized using antibodies

Question 4

A DNA molecule capable of replication in a host and into which a gene or piece of DNA can be specifically inserted at known positions

Answer 4

Cloning vector

Question 5

Cloning Vector Steps

Answer 5

1. Cut plasmid and target DNA with a restriction endonuclease that leaves sticky ends
2. Mix plasmid and target DNA to allow sticky ends to connect
3. Complete with ligase

Question 6

Plasmid Cloning Vector Requirements

Answer 6

1. Ori sequence
2. Dominant selectable marker (e.g. antibiotic resistance)
3. Unique restriction site inside a reporter gene
4. A reporter gene (LacZ: blue when functional and exposed to X-Gal)

Question 7

Lambda Cloning Vector

Answer 7

Engineered phage with only lytic capabilities that can insert DNA into bacteria

Question 8

Cosmid Cloning Vectors

Answer 8

Engineered to combine phage and plasmid features

1. Ori sequence
2. Dominant selectable marker
3. Unique restriction sites

Question 9

BAC

Answer 9

Bacterial Artificial Chromosome: Remove all “production” DNA so that new DNA can be inserted and all nutrients can be added to media instead of produced

Question 10

YAC

Answer 10

Yeast Artificial Chromosome: Used for cloning long genes

1. Centromere
2. Two telomeres
3. Origin of replication
4. Restriction sites to insert DNA

Question 11

PCR

Answer 11

Polymerase Chain Reaction

• DNA is denatured (melted) at 95*C
• Primers anneal at 55*C
• Taq polymerase elongates strand at 70*C

Question 12

VNTR

Answer 12

Variable Number of Tandem Repeats

• Repeated sequences in the genome that vary between people
• Can match tissue by visualized exact same repeat numbers
• Can match inheritance by having one band from each parent