Chapter 20

Question 1

Restriction endonucleases

Answer 1

enzymes that cut a DNA molecule at a particular place

Question 2

DNA ligase

Answer 2

seals nicks in the DNA backbone, covalently bonding the two strands

Question 3

Vectors

Answer 3

• Carrier DNA molecules

- Carry selectable gene marker to distinguish cells that have taken them up from those that have not

Question 4

Transformation - purpose

Answer 4

To introduce plasmids into bacteria

Question 5

Transformation - 2 main techniques

Answer 5

1. Heat shock

2. Electroporation (electricity)

Question 6

BACs

Answer 6

• Bacterial Artificial Chromosomes

- Generally very large but low copy number of plasmids

Question 7

YACs

Answer 7

• Yeast Artificial Chromosomes

- have telomeres at each end, ORI, and centromere

Question 8

Expression Vectors

Answer 8

• Designed to ensure mRNA expression of cloned gene

Question 9

DNA Libraries - 2 types

Answer 9

1. Genomic

2. Complementary (cDNA)

Question 10

Genomic Library

Answer 10

Contains at least one copy of all sequences in genome of interest

Question 11

cDNA library

Answer 11

contains complementary DNA copies made from mRNAs present in the cell’s population

Question 12

Reverse transcriptase - function

Answer 12

Generates cDNA from mRNA

Question 13

What does reverse transcriptase produce?

Answer 13

mRNA-DNA double-stranded hybrid molecule

Question 14

What do probes do?

Answer 14

sort through library and isolate specific genes of interest

Question 15

What is a probe?

Answer 15

any DNA or RNA sequence complementary to the target gene of sequence being identified

Question 16

PCR - 4 requirements

Answer 16

• DNA Polymerase
• DNA Template
• Primers
• Salt (such as magnesium)

Question 17

PCR - 3 steps

Answer 17

1. Heat up to denature
2. Primers anneal to the template
3. Primer extension

Question 18

Southern blot - 5 steps

Answer 18

1. DNA samples that are cut with restriction enzymes are loaded on agarose gel for electrophoresis
2. DNA is separated by electrophoresis
3. DNA-binding filter… placed on gel
4. Filter placed in heat-sealed bag/tube with solution containing labeled probe
5. Bound probe detected by film or probe signal capture with a digital camera

Question 19

Southern vs Northern vs Western blot

Answer 19

Southern: DNA-DNA hybridization
Northern: RNA-DNA hybridization
Western: Proteins

Question 20

Agrobacterium-mediated plant transformation

Answer 20

bacterial chromosome is put into Ti plasmid

Question 21

Particle bombardment plant transformation

Answer 21

• coat DNA in gold
• place into tubing
• cut tubing into 1-2 mm length
• put in chamber, connect to high pressure can, and use pressure to bombard the sample

Question 22

Using Embryonic Stem cells, scientists introduce targeting vectors into cells via _____.

Answer 22

Electroporation