Lecture 14 - DNA techniques

Question 1

How does gene organization differ from prokaryotes and eukaryotes?

Answer 1

Prokaryotes: multiple protein-coding genes are encoded in an operon, under the control of one set of regulatory elements.
Eukaryotes: each gene has its own set of regulatory sequences, which are bound by proteins that regulate expression of that gene.

Question 2

What must cloning vectors be capable of? Name 2 examples of cloning vectors

Answer 2

• Cloning vectors must be capable of self-replication

- Plasmids and Viral DNA

Question 3

What are restriction endonucleases (RE)?

Answer 3

Restriction endonucleases are bacterial enzymes that recognize and cleave DNA at specific sequences.

Question 4

What does “Restriction enzymes are often homodimers” mean?

Answer 4

2 identical enzymes bind to the same site of the DNA - one cuts each strand.

Question 5

When restriction enzyme hydrolyze a phosphodiester bond within a DNA strand, what happens?

Answer 5

This leaves a 5’-PO4 on one side of the cut, and a 3’-OH at the other side for each strand.

Question 6

What happens when restriction enzymes cut right in the middle of a recognition sequence?

Answer 6

It produces blunt ends

Question 7

What happens when the restriction enzymes cut outside of the centre of the recognition sequence?

Answer 7

It creates “sticky ends” or “overhangs.” They can either be 3’-overhangs or 5’-overhangs.

Question 8

What is the recognition sequence for EcoRI? Does it leave blunt ends or an overhang?

Answer 8

G/AATTC: 5’-overhang

Question 9

Why are REs that produce blunt-ends not as efficient?

Answer 9

Because the complementary strands cannot anneal (join by base pairs), although they can still be ligated together.

Question 10

What is SDM?

Answer 10

Site-directed mutagenesis: changes the nucleotide sequence of the cloned gene at a specific site, which changes the amino acid sequence of the gene product

Question 11

What are the 2 different approaches of SDM?

Answer 11

• Site-directed mutagenesis

- Oligonucleotide-directed mutagensis

Question 12

What does BAC stand for?

Answer 12

Bacterial Artificial Chromosomes

Question 13

What happens when bacterial DNA is methylated by something like methyltransferase?

Answer 13

The methylation identifies the DNA as bacterial DNA - will not get cleaved by bacterial RE, whereas non-methylated foreign DNA will be degraded.

Question 14

Restriction sequences are normally how long?

Answer 14

4-6 base pairs (bp) long

Question 15

What does DNA ligase require?

Answer 15

ATP, a 3’-OH end, and a 5’-PO4 end

Question 16

What is the Sanger dideoxy method?

Answer 16

DNA sequencing by controlled termination of replication. Uses electrophoresis to separate DNA strands differing by just a single nucleotide.

Question 17

For Sanger DNA sequencing, what does each of the 4 rxn tubes contain?

Answer 17

1. Normal deoxyribonucleoside triphosphate precursors (dATP, dCTP, dGTP, dTTP)
2. Oligonucleotide primer for DNA polymerase
3. Small amount of one dideoxyribonucleoside triphosphate (ddATP)

Question 18

How are products identified for Sanger DNA sequencing?

Answer 18

Each ddNTP has a unique fluorescent label attached, and products are identified using a detector. They used to have radioactive primers.

Question 19

What is PCR?

Answer 19

Polymerase chain reaction: it “amplifies” specific DNA sequences in vitro.

• extremely fast, extremely sensitive + requires very little template DNA.
• the amplified DNA can be sequenced or cloned into a vector

Question 20

What are the 3 steps of PCR?

Answer 20

1. heat to separate strands
2. hybridization of primers
3. DNA synthesis from primers

Question 21

With PCR, how many double stranded molecules are produced from cycle 1, 2, 3, 4 and

Answer 21

Cycle 1: 2
Cycle 2: 4
Cycle 3: 8
Cycle 4: 16
Cycle 5: 32

Question 22

What is PCR good for?

Answer 22

• isolation of a known fragment from complex genome or from the genomes of many individuals
• cloning of a gene/gene fragment for protein expression
• forensics
• study of relatedness
• studies of molecular evolution
• diagnostic tool for particular virus of bacteria
• isolation of multigene families

Question 23

What is a DNA library?

Answer 23

A collection of all the DNA fragments, cloned into vectors and stored in bacteria

Question 24

What is a genomic library?

Answer 24

A genomic library is generated by cleaving the entire genome of an organism into thousands of fragments and inserting each fragment into its own cloning vector.