Lecture 5: Recombinant DNA technology and DNA cloning Flashcards

1
Q

Recombinant DNA

A

DNA molecule made in vitro with segments from different sources

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2
Q

DNA Cloning

A

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

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3
Q

Genetic Engineering

A

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

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4
Q

Plasmids

A
  • 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

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5
Q

Phage

A

A virus that infects and replicates within a bacterium

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6
Q

Restriction Enzyme

A

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

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7
Q

Palindrome

A

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

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8
Q

Types of Enzyme

A

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

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9
Q

DNA Ligase

A

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

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10
Q

Chemical Transformation

A

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.

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11
Q

Chemical Transformation

A

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.

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12
Q

Electroporation

A

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

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