Chapter 11- Genetic Engineering and Biotechnology

Question 1

Genetic Engeneering

Answer 1

refers to the use of in vitro techniques to alter genes in he labratory

Question 2

restriction enzymes

Answer 2

recognize specific base sequences within DNA and cut the phosphodiester backbone, resulting in double-stranded breaks

they are used for in vitro DNA manipulation and are major tool of genetic engineering

Question 3

which species are restriction enzymes in

Answer 3

widespread among both Bacteria and Archaea (prokaryotes) and are very rare in eukaryotes

Question 4

what do restriction enzymes protect prokaryotes from?

Answer 4

hostile foreign DNA such as virus genomes

Question 5

Type 1 restriction enzymes

Answer 5

• 1st to be discovered
• bind to DNA at their recognition sequence but cut the DNA at some random distance away.
• little practical value as they do not produce discrete restriction fragments or distinct gel-banding patterns

Question 6

Type 3 restriction enzymes

Answer 6

• bond to the DNA at their recognition sequence but cut the DNA outside of their recognition sequence (~ 25 bp away from site)
• require two sequences in opposite orientations within the same DNA molecule to accomplish cleavage
• large combination restriction and modification enzymes

Question 7

Type 2 restriction enzymes

Answer 7

• cleave DNA within their recognition sequence; most useful for specific DNA manipulation
• recognize inverted repeat sequences (palindromes)

Question 8

palindrom

Answer 8

a nucleic acid sequence that is the same whether read 5’ to 3’ on one strand or 5’ to 3’ on the complementary strand with which it forms a double helix

Question 9

EcoR1

Answer 9

A restriction enzyme isolated from strains of E.coli, and is part of the restriction modification system
- Created 4 nucleotide sticky ends with 5’ end overhangs of AATT

Question 10

Nucleic acid sequence where the enzyme,EcoR1, cuts

Answer 10

GAATTC

complementary sequence is CTTAAG

Question 11

Modification Enzymes

Answer 11

Protect cell’s DNA from its own (or other) restriction enzymes

• each type of restriction enzyme has separate modification enzymes
• chemically modify nucleotides in restriction recognition sequence (generally consists of methylation of DNA)

Question 12

Gel Electrophoresis

Answer 12

• separates DNA molecules based on size
• uses an electric field to separate charged molecules
• nucleic acids migrate through the gel toward the positive electrode due to their negatively charged phosphate groups

Question 13

The same DNA cut with different restriction enzymes will have ______ binding patters on an agarose gel

Answer 13

different

Question 14

Synthetic DNA

Answer 14

used for primers and probes and in site-directed mutagenesis

Question 15

Steps in DNA Amplification by PCR

Answer 15

(DNA is replicated in a test tube (DNA amplification))
- 1. Template DNA is denatured by heating
-2. Add a synthetic piece of DNA (oligonucleotides) flanking sequence of interest to the reaction mixture
- 3. Add DNA polymerase
- 4. Heat and cool
repeat many similar cycles

Question 16

Applications for PCR

Answer 16

• obtain DNA for cloning
• comparative studies to amplify genes and DNA sequences
• used in microbial ecology for bacterial ID
• amplify VERY small amounts of DNA (forensic purposes, diagnostic, ancient DNA)

Question 17

Variations of PCR

Answer 17

• reverse transcriptase PCR- can be used to make DNA from an mRNA template
• Real time PCR

Question 18

Nucleic Acid Hybridization

Answer 18

Base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix

Question 19

nucleic acid probe

Answer 19

segment of single stranded DNA that is used in hybridization and has a predetermines identity

Question 20

Southern Blot

Answer 20

• a hybridization procedure where DNA is IN THE GEL and probe is RNA or DNA
• DNA is subjected to RE digestion and run on an agarose gel
• DNA is denatures and transferred to a synthetic membrane
• Labelled DNA probe is used to find complementary sequences by hybridization with a radioactive probe
• positions of hybridized bands noted using X-ray radiography
• only some of DNA fragments hybridize to probe

Question 21

Northern Blot

Answer 21

RNA IS IN THE GEL.

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

Question 22

FISH: Flourescent In Situ Hybridization

Answer 22

• uses fluorescent probe attached to oligonucleotide

Question 23

Molecular Cloning

Answer 23

Isolation and incorporation of a piece of foreign DNA into a vector so it can be replicated and manipulated

Question 24

Three main steps of gene cloning

Answer 24

1. Isolation and fragmentation of source DNA
2. Insertion of DNA fragment into cloning vector (DNA ligase)
3. Introduction of cloned DNA into host organism

Question 25

Key enzymes used for cloning

Answer 25

• restriction endonucleases
• DNA ligase
• Reverse transcriptase ( RNA –> DNA)
• DNA polymerase

Question 26

DNA ligase

Answer 26

Catalyzes the joining of two strands of DNA between the 5’-phosphate and 3’ OH of adjacent nucleotides with either cohesive or blunt ends

Question 27

DNA polymerase

Answer 27

Mostly used for 5’3’ polymerizing activity. May also have 3’5’ and 5’3’ exonuclease activity. The Klenow fragment lacks the later activity

Question 28

Plasmids

Answer 28

self replicating to amplify genes

• small size = easy to isolate DNA
• independent origin of replication

Question 29

Ideal Hosts for Cloning Vectors

Answer 29

• capable of rapid growth in inexpensive medium
• nonpathogenic
• capable of incorporating DNA
• Genetically stable in culture
• Equipped with appropriate enzymes to allow replication of the vector
  (E.coli, Bacillus subtilis, Saccharomyces cerevisiae)

Question 30

pUC19

Answer 30

a commone cloning vector

• has all essential elements of a cloning vector
• derived from ColE1 toxin-encoding plasmid
• lacZ genes
• ampicillin resistance
• polylinker (multiple cloning site) within lacZ gene

Question 31

Blue-white screening

Answer 31

In vectors with lacZ genes, the lacZ gene is inactivated by insertion of foreign DNA and B-galactosidase is NOT produced

• Therefore, Xgal, a substrate for B-galactosidase added to growth medium is not cleaved and a blue color does NOT develop
• BLUE colones do NOT have vector with foreign DNA inserted
• WHITE colones HAVE foreign DNA inserted

Question 32

Methods to Detect Clones containing CORRECT inserts

Answer 32

Initial selection- antibiotic resistance; blue-white screening

• Antibodies
• nucleic acid probes
• colony hybridization

Question 33

Reporter genes

Answer 33

encode proteins that are easy to visually detect and assay ( lacZ, luciferase, Green fluorescent Protein genes)

Question 34

Gene Fusions

Answer 34

Promoters of coding sequences of genes of interest can be swapped with those of reporter genes to elucidate gene regulation under various conditions

Question 35

Ligation Independent Cloning

Answer 35

does not require restriction enzymes or ligase

• depends on the 3’-5’ endonuclease activity of T4 DNA polymerase as well as its polymerase activity
• Vector and insert will have 4 nicks that are repaired by E.coli

Question 36

Site-directed mutagensis

Answer 36

performed in vitro and introduces mutations at a precise location
-can be used to assess the activity of specific amino acids in a protein

Question 37

Basic Procedure of Site-directed Mutagenesis using synthetic DNA

Answer 37

1. clone into single stranded vector
2. Add synthetic oligonucleotide with one base mismatch
3. Extend single strand with DNA polymerase
4. Transformation and selection

Question 38

Cassette mutagensis

Answer 38

DNA fragment can be cut, excised and replaced by a synthetic DNA fragment

Question 39

Gene distribution

Answer 39

when cassettes are inserted into a gene disrupting its function (insertional inactivation)

Question 40

knockout mutation

Answer 40

total loss of gene function

-gene disruption/insertional inactivation my cause this