Recombinant DNA Technology And Biotechnology 11/15

Question 1

Important achievements leading to modern molecular biology

Answer 1

• restriction endonuclease
• cloning of DNA
• creation of synthetic probes (paternity test)
• PCR

Question 2

Enzymes that cleave very specific DNA sequences

Answer 2

Restriction endonuclease

Question 3

What kind of sequences are recognized by restriction endonuclease

Answer 3

• Generally short (4-8 bp)

- palindromes

Question 4

DNA palindromes

Answer 4

Read the same 5’ to 3’ on both strands

Question 5

Is this a palindrome?
5’ TCTAGA 3’
3’ AGATCT 5’

Answer 5

Yes

Question 6

Is this a palindrome?
5’ TCTTCT 3’
3’ AGAAGA 5’

Answer 6

No

Question 7

What kind of ends to restriction enzymes cleave to leave?

Answer 7

Sticky or blunt ends

Question 8

Sticky ends

Answer 8

• staggered or cohesive

- allows the 2 nucleotide base pair. Helps restriction enzyme. More favorable

Question 9

Blunt ends

Answer 9

-no orientation regulation, less favorable. Just a clean straight down cut

Question 10

What is attached after cleavage done by the restriction enzyme?

Answer 10

The 3’ OH group and the 5’ phosphate are attached after cleavage (important for ligation) for both sticky and blunt ends

Question 11

How are restriction enzymes named?

Answer 11

Generally for the organisms they were derived from

Question 12

DNA sequences that can be cleaved by a restriction enzyme

Answer 12

Restriction site.

Question 13

Restriction enzymes that recognize larger sequences…

Answer 13

Cut less frequently

Question 14

Restriction enzymes that recognize shorter sequences,

Answer 14

Cut more frequently

Question 15

Tools to cut, paste, and analyze DNA

Answer 15

Restriction enzymes

Question 16

Recombinant DNA

Answer 16

Fragments of DNA can be “pasted” together to make hybrid molecules

Question 17

What kind of ends are better for recombinant DNA?

Answer 17

Sticky ends

Question 18

Enzyme which creates the phosphodiester bonds

Answer 18

DNA ligase

Question 19

Inserting a restriction fragment (recombinant DNA) into a cloning vector

Answer 19

DNA cloning

Question 20

What happens to the vector in host cells?

Answer 20

Can then be replicated

Question 21

inserting a restriction fragment into a cloning vector, vector replicated in host cells, and DNA now cloned and amplified

Answer 21

Recombinant DNA amplification

Question 22

Molecules of DNA that can accept fragments of foreign DNA

Answer 22

Vectors

Question 23

Vector requirements

Answer 23

• must be capable of autonomous replication in the cell
• must have at least one restriction site for foreign DNA insertion
• must carry at last one gene for selection (usually antibiotic resistance)

Question 24

What is the most common vector

Answer 24

Prokaryotic (bacterial) plasmids

Question 25

What is the second most common vector

Answer 25

Yeast

Question 26

Two types of DNA libraries

Answer 26

• genomic DNA libraries

- cDNA libraries

Question 27

Genomic DNA libraries

Answer 27

• entire genome chopped up with restriction enzymes, cloned in vectors, and used to transform bacteria
• each transformed bacteria containting a plasmid may contain a different segment of the genome
• collection contains ALL SEQUENCES IN THE GENOME, including coding regions as well as introns and other intervening sequences

Question 28

CDNA library (complimentary DNA)

Answer 28

• cDNA generated using isolated mRNA from a particular cell or tissue type
• mRNA is reverse transcribed (by reverse transcriptase) and the second strand is synthesized using a DNA polymerase
• cDNA is ligated into a vector, used to transform bacteria and 1000s of clones are collected

Question 29

Ideal cDNA library

Answer 29

Will contain sequences representing all mRNAs present in the cell or tissue types at the time the mRNA was collected

Question 30

CDNA libraries allow

Answer 30

• allow one to see what genes were being expressed in a particualr cell or tissue type
• only contain mRNA sequences ( no introns, promotes)