Genetic Engineering

Question 1

Human manipulation of an organism’s DNA

Answer 1

Genetic Engineering

Question 2

Current Methods of Genetic Engineering include recombinant DNA technology (rDNA):

Answer 2

transplanting a gene (trans gene) from one organism to another

• ex. insulin production
• created using molecular cloning

Question 3

DNA of interest is inserted into a cloning vector.

Answer 3

Molecular Clone

Question 4

What would you use the final clone for? What kind of experiments would you use it in?

Answer 4

• Use the gene product
• Figure out what gene product does:
  
  • test the biochemistry
  • use a model organism
  • localize protein with a tag
• Diagnoses of disease, genotype
• Gene therapy
• Make a mutation
• Sequence gene of interest

Question 5

• a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.
• contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together.

Answer 5

Cloning Vector

Question 6

General Cloning Strategy

Answer 6

1. Isolate DNA of interest from an organism
2. Cut the DNA with a restriction enzyme
3. Cut the cloning vector with the same restriction enzyme
4. Ligate piece into a cloning vector to make a recombinant DNA molecule
5. Transform the recombinant DNA into a host (like E. coli), the host cell will make identical copies called clones.

Question 7

Products of restriction enzymes:

Answer 7

Sticky Ends

Question 8

Relationship between restriction enzymes and ligase:

Answer 8

Opposing roles; Restriction enzymes cut and Ligase seals

Question 9

A collection of plasmids (clones) in which each plasmid theoretically contains a different piece of DNA.

Answer 9

Genomic Library

Question 10

General Steps to make a Genomic Library

Answer 10

1. DNA gets extracted from an organism of interest
2. DNA is then cut up into small pieces using restriction enzymes
3. Each piece gets ligated into cloning vectors
4. Library is formed

Question 11

Importance of Genomic Libraries

Answer 11

• Can locate a specific sequence
• Assists with diagnostics
• Sequences by “shot-gunning”:
  
  • Screens, ie. suppressor screening by transforming a library into a mutant and checking for revertants to the wild type phenotype.

Question 12

Goal: to find a DNA sequence in a pool of DNA

-Use a DNA probe and Caveat

Answer 12

Screening a Genomic Library

Question 13

a labeled segment of DNA or RNA used to find a specific sequence of nucleotides in a DNA molecule.
-may be synthesized in the laboratory, with a sequence complementary to the target DNA sequence.

Answer 13

DNA probe

Question 14

General Steps in making a cDNA Library

Answer 14

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

Question 15

Importance of cDNA Librares

Answer 15

• Detects sequence of interest
• Allows you to tell the difference between exons and introns
• Can look for splice variance (Alternative Splicing)

Question 16

It is DNA synthesized from a mRNA template in a reaction catalysed by the enzymes reverse transcriptase.
-often used to clone eukaryotic genes in prokaryotes.

Answer 16

cDNA

Question 17

Amplify a DNA segment of interest.

-example: amplify a portion of DNA to clone into a cloning vector.

Answer 17

PCR–polymerase chain reaction

Question 18

Uses for PCR

Answer 18

• Forensics
• Organ Matching
• Sequence an entire genome/transcriptome
• quantify a transcript
• Make mutations in a gene of interest
  
  • M vs. F
  • ++ vs. Mutant
  • Diseased vs. Healthy

Question 19

Measures the increase in the amount of PCR product during the thermal cycling/rxn.

• Measure a specific DNA as it’s amplified.
• Therefore, can measure abundance of transcript in a sample.

Answer 19

Real-time PCR aka Quantitative PCR (qPCR)

Question 20

Reasons for mutagenesis:

Answer 20

• Suppresor mutations (“fix it”)
• Study the mutation: see if it’s advantageous, identify a disease-causing mutation, identify the “important” amino acid in a polypeptide to understand the role of a particular amino acid in a polypeptide.

Question 21

2 Major types of mutagenesis; ex) they add Mn+2 instead of Mg+2, which causes Polymerase to have reduced fidelity (will add mistakes)

Answer 21

1. Site Directed Mutagenesis

2. Random Mutagenesis

Question 22

Intentionally mutate 1 specific nucelotide.

-include primer with single nucleotide change.

Answer 22

Site Directed Mutagenesis

Question 23

Create random mutations in DNA of interest

Answer 23

Random Mutagenesis

Question 24

Overall goal: To detect a specific DNA sequence in a sample of DNA

• Used to:
• compare homologous genes
• analyze intron organization
• paternity tests
• diagnostics of diseases
• look for methylated sequences

Answer 24

Molecular Analysis of DNA by Southern Blotting

Question 25

General Steps of Southern Blotting:

Answer 25

1. Isolate DNA from an organism of interest.
2. Cut DNA using fragments using restriction enzymes
3. Separate DNA fragments with gel electrophoresis
4. Transfer DNA from gel to a membrane (“blot”)
   * DNA will end up in the same position as it appeared on gel.
5. Incubate blot with labeled probe complementary to DNA of interest.
6. Visualize probe-band with DNA of interest

Question 26

Example of Southern Blotting: RFLP diagnosis of Sickle Cell Anemia

Answer 26

See notes.

Question 27

Uses of DNA polymorphisms in genetic analysis

Answer 27

• ex. SNPs

- Differ by nucleotide sequence or number of tandemly repeated nucleotide units.