12- Chapter 12

Question 1

What is genetic engineering?

Answer 1

Using in vitro techniques to alter genetic material
Recombinant DNA technology is artificial recombination of DNA from two organisms
Slide 4 Oct 26

Question 2

What are Restriction Endonuclease Enzymes (REases)?

How are they designated (named)?

Answer 2

Recognize specific DNA sequences and cut DNA
Rare in eukaryotes, not prokaryotes
Protects from hostile foreign DNA by destroying it
They are designated by 3 letters
1st- represents genus
2nd and 3rd- species RE isolated from

Question 3

What are 3 classes of Restriction Enzyme Endonucleases?

Answer 3

Type I- cuts DNA at random far from recognition sequence (no practical value)
Type II- cleaves DNA within recognition sequence (most useful for DNA manipulation)
Structure of cleavage may differ (3’ or 5’ overhang or blunt ends)
Type III- large combo of restriction and modification enzymes
Cleaves outside of recognition sequence
Slide 7 oct 26
Slide 10 oct 26

Question 4

What are palindromes?

Answer 4

Invert repeated sequences
Type 2 REases recognize these
Nucleic acid sequence that is same whether it’s read 5’ to 3’ on one strand or 5’ to 3’ on the complementary strand
GAATTC
CTTAAG
Palindrome words: civic dad madam Race car

Question 5

What is Eco R1? (A type II REase)

Answer 5

Restriction endonuclease enzyme from E. coli
Creates 4 nucleotide sticky ends with complementary 5’ over hangs
Cuts at the nucleus acid sequence GAATC
Complementary is CTTAG
Slide 12 oct 26

Question 6

What is molecular cloning and it’s 3 steps?

Answer 6

Isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated
Plasmids are
1. Isolation and fragmentation of source DNA
2. Insertion of DNA fragment into cloning vector
3. Introduction of cloned DNA into host organism
Slide 14 oct 26

Question 7

What are the 4 enzymes used for cloning?

Answer 7

Restriction endonucleases
DNA Ligase- joins two strands of DNA
Reverse transcriptase- coverts RNA to DNA
DNA polymerase- used for 5’3’ polynerizing activity
Sometimes 3’5’ and 5’3’ exonuclease activity

Question 8

How are clones selected by insertions activation?

What do white and blue colonies mean (blue white screening)

Answer 8

In pUC19 or other vectors with lacZ genes, lacZ is inactivated by insertion of foreign DNA and β-galactosidase is not produced
Then Xgal (substrate for β-galactosidase) added to growth medium is not cleaved and blue colour does not develop for blue white screening
Blue means no vector with foreign DNA
White means foreign DNA inserted
Slide 19 oct 26

Question 9

What is a genomic library?

How do you clone only expressed genes?

Answer 9

Nearly complete or complete copy of an organism ms genome contained as recombinant DNA plasmids or phages (larger DNA fragments are clones into phage vectors)
To clone only expressed genes libraries are made using organisms mRNA
mRNA cannot be clones directly

Question 10

What are the characteristics for ideal hosts for cloning?

Answer 10

Capable of rapid growth
nonpathogenic
Capable of incorporating DNA
Genetically stable 
Has proper enzymes for relocation of vector
(E. Coli, Bacillus subtilis, etc)

Question 11

What are the 3 ways recombinant molecules are introduced into eukaryotic hosts?

Answer 11

Electroporation
Microinjection
Gene gun
Slide 24 oct 26

Question 12

What is electrophoresis (DNA ladder)?

Answer 12

Use of electrical field to separate charges molecules
Nucleic acids go through gel to positive end since negatively charged
Same DNA cut with different restriction enzymes will have different binding patterns on agarose gel
Slides 25-27 oct 26

Question 13

What is DNA amplification and Polymerase Chain Reaction (4 steps) (PCR)?

Answer 13

DNA amplification is DNA replication in a test tube to clone specific stuff or determine DNA sequences in regions or paternity determination or crime etc
PCR first denatures the amplified DNA by heating
Then synthetic piece of DNA of interest is added
Then DNA polymerase is added
Then heated and cooled
Slide 29-30 oct 26

Question 14

What is nucleic acid hybridization?

What is a nucleic acid probe?

Answer 14

Base luring if single strands of DNA or RNA from two different sources to give a hybrid double helix
Nucleic acid probe is a segment of single strand DNA that is used in hybridization with predetermined identity
Slide 36 oct 26

Question 15

What are four perks of using synthetic DNA?

What is FISH?

Answer 15

Commercial instruments available for de novo synthesis of DNA
Oligonucleotides of 100 bases can be made
Multiple oligonucleotides can be ligated (joined) together
Synthesized DNA is used for primers and probes
FISH- Fluorescent In Situ Hybridization
Use of fluorescent probe attacked to oligonucleotide
Slide 38 oct 26

Question 16

What is a southern blot?

Answer 16

A hybridization procedure where DNA is in gel and probe is RNA or DNA
Positions of hybridized bands noted using X-day radiography or fluorescence
Slide 40 oct 26

Question 17

What are northern blots?

Answer 17

Hybridization procedure where RNA is in the gel

Radioactive probe to a specific gene to total RNA which is run on a gel

Question 18

How do you detect clones containing correct DNA inserts?

Answer 18

See if cloning procedure worked
Initial selection- antibiotic resistance and blue white screening
If cells express foreign gene (expression can be detected by antibodies)
Looks for specific DNA if the gene is not expressed
Slide 44 oct 26

Question 19

What are the cloning tools reporter genes and gene fusions?

Answer 19

Reporter genes- encode proteins that are easy to visually detect and assay (lacZ. green fluorescent protein)
Gene fusions- promoters or coding sequences of genes of interest can be swapped with those of reporter genes to elucidate gene regulation
Slide 46 oct 26

Question 20

What is site-directed mutagenesis?

Answer 20

Performed in vitro and introduces mutations at a PRECISE location
Can be used to assess the activity of specific amino acids
Slide 49 oct 26

Question 21

What is cassette mutagenesis?

Otherwise known as insertions inactivation

Answer 21

Cassettes (after antibiotic resistance genes) are inserted into a gene, disrupting its function
The result is a knockout mutation
Slide 51-55 (55) oct 26

Question 22

What is a knockout mutation?

Answer 22

Total loss of gene function
Happens when disrupted gene from cassette mutagenesis is recombined into the chromosome
Slides 51-55 (55) oct 26