Lecture 5: Recombinant DNA technology and DNA cloning

Question 1

Recombinant DNA

Answer 1

DNA molecule made in vitro with segments from different sources

Question 2

DNA Cloning

Answer 2

Generation of multiple identical copies of a gene or defined DNA segment for further study

Question 3

Genetic Engineering

Answer 3

Using recombinant DNA technology to manipulate genes for practical purposes including developing products

Question 4

Plasmids

Answer 4

• A small DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently
• Average size 1000-4000 base pairs
• Extrachromosomal DNA: they are separate from the bacterial chromosomal DNA
• Self-replicate: have a single origin of replication

Advantages
- plasmids exist in high copies, are isolated easily from bacteria, and can be manipulated easily in vitro to insert foreign DNA, to create recombinant plasmids
- plasmids can be taken up readily by bacteria in the process of transformation, to generate recombinant bacteria

Common Features
- antibiotic selection marker: selection of transformed cells
- ori: the origin of replication
- lacZ: screening of transformed cells containing recombinant DNA
- Multiple cloning site (MCS): contains many unique restriction enzymes sites for insertion of foreign DNA
- Transcription promoter (place): expression of recombinant DNA

Question 5

Phage

Answer 5

A virus that infects and replicates within a bacterium

Question 6

Restriction Enzyme

Answer 6

Are enzymes that recognize a short (4-8 nt) specific sequence of nucleotides (a palindrome) on DNA and cuts the DNA at specific places within the palindrome
- Restriction enzyme sites usually occur many times in a long DNA molecule- a given enzyme should make many cuts in a very long DNA molecule

Question 7

Palindrome

Answer 7

A DNA sequence that is read the same whether rending in the 5’ to 3’ on one stand to 5’ to 3’ on the complementary strand of the double helix

Question 8

Types of Enzyme

Answer 8

Type I: require both ATP and S-adenosyl-L-methionine
Type 2: requires magnesium
Type 3: short distance from a recognition site
Type 4: target modified DNA
Target 5: utilize guide RNAs

Question 9

DNA Ligase

Answer 9

Is an enzyme that covalently repairs the phosphodiester bond between 3’ OH end of one fragment and 5’ PO4 end of another DNA fragment to join restriction fragments together

Question 10

Chemical Transformation

Answer 10

Bacteria cells are treated with ice-cold calcium chloride. Plasmid is then added to chilled cells where Ca2+ coats the PO3-. The cell and DNA mixture is heated to 42C (heat shock) and the membrane is damaged and plasmid DNA is able to enter bacterial cells where it replicates.

Question 11

Chemical Transformation

Answer 11

Bacteria cells are treated with ice-cold calcium chloride. Plasmid is then added to chilled cells where Ca2+ coats the PO3-. The cell and DNA mixture is heated to 42C (heat shock) and the membrane is damaged and plasmid DNA is able to enter bacterial cells where it replicates.

Question 12

Electroporation

Answer 12

Chilled cells are subjected to electric pulse and bacterial membranes open up temporarily and allows DNA to go in