Chapter 7: 7.1 Basics of Genetic Engineering

Question 1

What does genetic engineering allow us to identify?

Answer 1

THe role of every amino acid in a protein

Question 2

What is genetic engineering used to study?

Answer 2

Protein structure and function

Question 3

Genetic manipulation

Answer 3

Select genes can be input (“cloned”) into plasmids either in their native form or manipulated/mutated form for genetic manipulation or protein analysis

Question 4

Genes can be cloned from any ——- DNA

Answer 4

Genomic

Question 5

Can cloned genes be manipulated?

Answer 5

Yes

Question 6

What are the 4 steps in cloning?

Answer 6

1. Clone gene
2. Insert cloned gene into plasmid
3. Insert plasmid into host
4. Grow host

Question 7

Cloning:

What can the gene be cloned from?

Answer 7

• cDNA
• mRNA
• Genomic DNA (prokaryotes only)

Question 8

What are expression vectors in cloning?

Answer 8

Plasmids

Question 9

Plasmids

Answer 9

• Circular form of dsDNA
• Often are/are derived from naturally occuring bacterial DNA

Question 10

The insertion of a gene of interest into plasmid results in…

Answer 10

Recombinant DNA
* DNA consisting of genetic material from several sources

Question 11

What are the key components of plasmids?

Answer 11

• Origin of replication
• Selection marker
• Multiple cloning site
• Promoter

Question 12

What are circularized plasmids replicated in bacteria for?

Answer 12

To produce more:
* Plasmid
* Protein (translated from gene insert)

Question 13

Restriction enzymes

Answer 13

Restriction endonuclease (RE)
* Cut dsDNA at palindromic sites

Question 14

What did restriction endonucleases come from?

Answer 14

Prokaryotic cells

Question 15

What types of ends can restriction enzymes create?

Answer 15

• Blunt ends
• Sticky ends

Question 16

What must be introduced to the ends of your gene of interest if you want to insert it into a plasmid?

Answer 16

Introduce RE sites to ends of gene
* Done by PCR

Question 17

The RE sites at the end of your gene of interest must match…

Answer 17

Sites in the plasmid of interest

Question 18

After RE cuts the gene of interest and the plasmid, what fuses them together?

Answer 18

DNA ligase fuses the complementary sequences together

Question 19

PCR

Answer 19

Polymerase Chain Reaction
* Used to amplify and alter genetic material using short complementary oligos called primers

Question 20

What are the steps in PCR?

Answer 20

1. Denaturation
2. Annealing
3. Elongation
4. Repeat

Question 21

Steps in PCR:

Denaturation

Answer 21

Double-stranded DNA is separated at high temperatures

Question 22

Steps in PCR:

Annealing

Answer 22

Primers bind to their complementary DNA at primer specific temperatures

Question 23

Steps in PCR:

Elongation

Answer 23

Nucleotide bases are added onto the ends of primers by thermostable DNA polymerase

Question 24

In PCR, how many genes are present after each cycle?

Answer 24

Double the number of genes