ADL Lecture 7: 16.2-4; 18.1

Question 1

Recombinant DNA technology

Answer 1

• set of techniques for amplifying, maintaining and manipulating DNA sequences (in vitro / vivo)
• divide genome into smaller segments to analyze/reassemble to provide molecular view of genes and genomes

Question 2

Restriction Enzymes

Answer 2

-Recognizes specific DNA sequence (~4-8 bp) at which it cuts both strands of the sugar-phoshate backbone of DNA

Question 3

Restriction-modification systems

Answer 3

methylate restriction enzyme recognition sequences in bacteria, to protect the bacterial sequences from digestion

Question 4

Sticky ends

Answer 4

single stranded segments produced by some restriction enzyme cuts and can base-pair with complementary sequences

Question 5

What are some features of Restriction enzymes?

Answer 5

• common in bacteria
• some leave blunt ends [with no single stranded (ss) overhangs]
• the # and size of fragments produced by digesting with a given restriction enzyme depends on the size of genome and the relative abundance of each nucleotide

Question 6

Digestion of a large genome

Answer 6

-yields a smear of many fragments of differing sizes

Question 7

Molecular cloning

Answer 7

• After a genome has reduced to smaller pieces for study, pieces must be reproduced in large amounts to be studied further
• Isolated DNA fragments are inserted into a vector

Question 8

Vector

Answer 8

A DNA fragments with attributes that will allow its amplification (origin of replication) in a biological system and serves as a carrier for foreign DNA inserted into it. Vectors usually also possess genes (e.g. encoding resistance to an antibiotic) that allow selection of hosts carrying the vector

Question 9

DNA clones

Answer 9

A fragment of DNA that is inserted into a vector such as a plasmid, cosmid, of artificial

Question 10

DNA ligase

Answer 10

used to seal fragments from DNA molecules; the donor fragment becomes permanently joined in a combination that may never have existed before

Question 11

Recombinant Clone

Answer 11

a combination of DNA molecules from different sources (e.g. vector and insert DNA) that are joined together using recombinant DNA technology

Question 12

DNA transformation

Answer 12

1) bacterial process of gene transfer in which donated DNA fragments originating in a dad donor cell or plasmid DNA, is taken up across the cell wall and membrane of recipient cell and recombined into the transformant genome
2) process by which exogenous DNA is directly taken up by a cell resulting in a genetic alteration of the cell

Question 13

Detection of Transformed Cells

Answer 13

Use a plasmid that has an antibiotic-resistance marker and grow the transformed bacteria on a medium that contains the antibiotic: only cells hat contain plasmid can form a colony

Question 14

multiple cloning site (MCS)

Answer 14

A vector DNA sequence containing several unique restriction enzyme target sequences facilitating cloning of inserted DNA fragments

Question 15

What gene is MCS typically within and what does the insert prevent?

Answer 15

within the lacZ gene; prevents expression of lacZ

Question 16

What is the consequence of preventing expression of lacZ?

Answer 16

Beta-galactosidase cleaves the lactose analog X-gal and coverts it to a blue by-product; an insert prevents cleavage of X-gal and the colonies are white

Question 17

Vector: Plasmid
Form of DNA:
Capacity:
Uses:

Answer 17

circular
<15 kb
sub-cloning and cDNA libraries

Question 18

Vector: Lambda
Form of DNA:
Capacity:
Uses:

Answer 18

Linear phage chromosome
<23 kb
cDNA and genomic libraries

Question 19

Vector: Cosmid
Form of DNA:
Capacity:
Uses:

Answer 19

Circular
30-45 kb
Genomic libraries

Question 20

Vector: BAC
Form of DNA:
Capacity:
Uses:

Answer 20

bacterial chromosome
100-200 kb
genomic libraries

Question 21

Vector: YAC
Form of DNA:
Capacity:
Uses:

Answer 21

Yeast chromosome
250-2000 kb
Genomic libraries

Question 22

cDNA libraries

Answer 22

Collection of DNA clones, originally derived via reverse transcription of mRNA molecules into DNA (cDNA) and cloned into a vector

Question 23

Reverse transcriptase

Answer 23

Enzyme, derived from retroviruses or retrotransposons, that catalyzes the synthesis of a DNA strand (cDNA) from an RNA template