Recombinant DNA Technology

Question 1

recombinant DNA definition**

Answer 1

DNA molecule made in vitro (in lab/artificially) with segments (genetic material) from different sources

Question 2

plasmid

Answer 2

small circle of bacterial DNA that is independent of main bacterial chromosome
- can replicate independently

Question 3

restriction enzyme

Answer 3

a protein that cleaves DNA and produces DNA fragments

Question 4

DNA cloning

Answer 4

generation of multiple identical copies of a gene/DNA segment

Question 5

genetic engineering

Answer 5

using recombinant DNA tech to manipulate genes for practical purposes

Question 6

DNA cloning steps**

Answer 6

1. construction of recombinant DNA
2. transformation of host cell by adding recombinant DNA
3. selection of recombinant clones

Question 7

cloning vector

Answer 7

modified DNA molecule capable of replication in a host organism
- common cloning vectors: plasmids, phage, BAC

Question 8

features of plasmids used as cloning vectors

Answer 8

antibiotic selection marker: selection of transformed cells
ori: origin of replication
lacZ: screening of transformed cells containing recombinant DNA
multiple cloning sites (MCS): contains many unique restriction enzyme sites for insertion of foreign DNA
transcription promoter (Plac): expression of recombinant DNA

Question 9

restriction enzyme (in-depth)**

Answer 9

enzymes that recognize a short (4-8 nt), specific sequence of nucleotides (a palindrome) on DNA –> tells enzyme where to bind and where to cut DNA
- sequence is always palindrome
- palindrome = DNA sequence read same way in 5’ to 3’ in 1 strand and 5’ to 3’ in complementary strand
- overhangs/sticky ends on cut sequences can base pair with any sequence cut by same enzyme –> how recombinant plasmids are made (cut plasmid vector –> cut DNA of choice w/ same enzyme –> produce same sticky ends and join together with DNA ligase)

Question 10

construction of recombinant plasmid

Answer 10

step 1 of DNA cloning
1. restriction enzyme cuts sugar-phosphate backbones
2. DNA fragment added from another molecule cut by same enzyme –> base pairing occurs (H-bond)
3. DNA ligase seals strands

Question 10

DNA ligase

Answer 10

enzyme that restores phosphodiester bond between 3’ OH end of 1 fragment and 5’ PO4 end of another DNA fragment to join restriction fragments together

Question 11

transformation**

Answer 11

step 2 of DNA cloning
- recombinant DNA added to bacteria/host cell by transformation: process where bacterial cell membrane is temporarily made more permeable
- 2 types of transformation
– 1. chemical transformation: bacterial cells treated with ice-cold calcium chloride –> plasmid added to chilled cells –> cell and DNA mixture heated (heat shock) –> membrane is damaged and plasmid DNA able to enter bacterial cells where it replicates
– 2. electroporation: chilled cells subjected to electrical pulse –> bacterial membranes open temporarily and allows DNA to go in

Question 12

selection of recombinant clones

Answer 12

plate transformed bacteria on solid nutrient agar medium containing ampicillin and X-gal
1. selection = antibiotic resistance marker: only bacteria that have taken up plasmid (which carries ampR gene) will grow and reproduce to form colonies –> doesn’t tell if gene of interest is on plasmid just tells you plasmid is there
2. screening = blue/white screening: lacZ gene encodes the enzyme B-galactosidase that catalyzes hydrolysis of X-gal to form a blue product
– insertion of foreign DNA in a restriction enzyme site in the MCS (multiple cloning sites) disrupts lacZ in plasmid –> no functional b-galactosidase –> no blue chemical formed = white colonies even in presence of X-gal

Question 13

good summary of rDNA (recombinant DNA) from construction to selection/screening

Answer 13

pic on lec 5 slide 29
1. plasmid DNA and foreign DNA both cut with same restriction enzyme
2. foreign DNA inserted into plasmid, where it inactivates lacZ gene
3. recombinant plasmid introduced into a bacterium, which becomes ampicillin resistant
4. all treated bacteria spread on nutrient agar plate containing ampicillin and X-gal, and incubated
5. white colonies that appear must contain foreign DNA; blue colonies must not contain foreign DNA

Question 14

what is cDNA?

Answer 14

complementary DNA: made by cloning DNA made in vitro (in lab) by reverse transcription (RNA strand used as template for synthesis of complementary DNA strand) of all the mRNA produced by a particular cell or tissue at some specific conditions (mature mRNA - introns removed)
- made when you need particular set of genes at specific conditions

Question 15

how do you make cDNA?

Answer 15

1. isolate mature mRNA,–> add reverse transcriptase, mRNA dNTPs, & polyT primer to tube
2. synthesis of complementary strand of DNA using mRNA as template
3. mRNA degraded by RNAse
4. DNA polymerase synthesizes 2nd DNA strand using hexamers as primers
5. cDNA is complete
6. Linkers w/ restriction sites are ligated to both ends of cDNA
7. cDNA and plasmid vectors are cut w/ same restriction enzyme and then glued using ligase
8. recombinant cDNA libraries then transformed into bacteria to obtain cDNA libraries (just collection of the cloned DNA sequences complementary to mRNA)

Question 16

genomic library vs cDNA library

Answer 16

genomic library: complete collection of cloned DNA fragments that constitutes entire genome of organism
cDNA library: represents part of genome (only specific subset of genes transcribed in mRNA in specific cells, tissues, or stages where mRNA extracted from)
- cDNA produced has no introns