Recombinant DNA Technology

Question 1

Recombinant DNA technology

Answer 1

Transplanting genes (trans genes) from 1 organism to another

Question 2

Recombinant DNA (rDNA)

Answer 2

Created using molecular cloning

Question 3

Cloning vector

Answer 3

Plasmid used in cloning
Contains Ori (origin of replication), MCS (multiple cloning site), and gene for antibiotic resistance

Question 4

Creating single gene clones

Answer 4

1. Isolate DNA of interest from an organism
2. Cut the DNA with a restriction enzyme to make sticky ends
3. Cut the cloning vector with the same restriction enzyme to create complementary sticky ends
4. Ligate DNA into cloning vector to make a recombinant DNA molecule
5. Transform recombinant DNA into host (ex- E. coli): host makes clones

Question 5

Restriction enzymes

Answer 5

Enzymes that cut phosphodiester bonds of DNA

Sequence specific

Question 6

Ligase

Answer 6

Enzyme that re-seals phosphodiester bonds between sequence and cloning vector

Question 7

Creating a genomic library

Answer 7

1. DNA is extracted from organism of interest
2. DNA is cut into small pieces using restriction enzymes
3. Pieces are ligated into a cloning vector
4. Form library: collection of clones/plasmids in which each plasmid contains a different piece of DNA

Question 8

Screening a genomic library

Answer 8

Use DNA probe that is complementary to sequence of interest and contains a detectable tag to find a specific sequence in a pool of DNA

Question 9

cDNA

Answer 9

Complementary DNA: complementary to RNA

Question 10

Creating a cDNA library

Answer 10

1. RNA is extracted from organism of interest
2. RNA is reverse transcribed into cDNA
3. Each cDNA is cloned into vector
4. Each library clone has a different cDNA

Question 11

How to tell which sequences are exons vs. introns

Answer 11

Compare genomic library to cDNA library: sequences in genomic library absent in cDNA library are introns

Question 12

PCR (polymerase chain reaction)

Answer 12

Amplify millions of copies of DNA molecule from very small starting portion of DNA
Many cycles of 3 steps: denature DNA using heat, anneal primers to DNA, extend strand using Taq polymerase

Question 13

Real-time PCR or quantitative PCR (qPCR)

Answer 13

Measure increase in amount of PCR product during thermal cycling reactions
Measure specific cDNA as it’s being amplified: use reporter probe

Question 14

Reporter probe in qPCR

Answer 14

Contains fluorescent marker and quencher
When quencher is close to fluorescent marker, no light is emitted
As polymerase makes strand, quencher is displaced from fluorescent marker, causing light to be emitted

Question 15

Site-directed mutagenesis

Answer 15

Mutating 1 specific nucleotide

Done using primer with single nucleotide change

Question 16

Random mutagenesis

Answer 16

Creating random mutations in DNA of interest

Adding Mn+2 instead of Mg+2 causes polymerase to have reduced accuracy

Question 17

Southern blotting steps

Answer 17

1. Isolate DNA from organism of interest
2. Cut DNA into small pieces using restriction enzymes
3. Separate DNA fragments using gel electrophoresis
4. Transfer DNA from gel to membrane/blot (DNA is in same position on blot as on gel)
5. Incubate blot with labeled probe that is complementary to DNA of interest
6. Visualize probe: see band on blot where DNA of interest is located

Question 18

DNA polymorphisms

Answer 18

DNA that differs from other DNA molecules in terms of nucleotide sequence or number of tandemly repeated nucleotide units