final exam

Question 1

recombinant DNA technology is used to:

Answer 1

1. fragment DNA into easily managed pieces, and purify the pieces
2. create many copies of DNA fragments of identical sequence
3. combine DNA fragments to construct recombinant DNA molecules
4. determine the exact sequence of specific DNA molecules
5. identify fragments of DNA containing complementary sequences
6. introduce specific DNA molecules into living organisms
7. assay the phenotypic effects of the introduced DNA

Question 2

recombinant DNA technology

Answer 2

a set of techniques for amplifying, maintaining, and manipulating DNA sequences in vitro and in vivo
> main approach is to identify specific DNA sequences and manipulate them in vitro
> researchers often divide genome into smaller segments to be analyzed and reassembled to provide a molecular view of genes and genomes

Question 3

roles of various miRNAs

Answer 3

by controlling gene expression they can be involved in almost any process within an organism
> can create knockout mutants or over-expression mutants to determine the role of each individual miRNA
> they have been linked to various human diseases

Question 4

cloning of plants

Answer 4

• many plants have capacity for asexual propagation, which can produce clones that are all genetically identical
• useful in agriculture, for propagating desirable heterozygotes w/o segregation of alleles

Question 5

cloning of animals

Answer 5

• do not readily propagate clonally in nature, with some exceptions
• some aphid species undergo multiple clonal, parthenogenetic generations in spring/summer followed by sexual reproduction in autumn
• most animal cells are not totipotent, so cloning animals is very complicated

Question 6

advantages of pharming

Answer 6

• makes complex human proteins
• makes large amount of protein
• less expensive
• clinically equivalent

Question 7

GMOs grown for food in the U.S.

Answer 7

• > 50% of corn crops
• > 75% of soybean and cotton
• most processed foods contain them
• 1% of farmland in Europe is used to grow GMOs
• absent from most processed foods

Question 8

benefits of GMOs

Answer 8

• insect resistance (many)
• round-up resistance (many)
• cold tolerance (strawberries)
• edible vaccines ( potatoes & rice)
• vitamin A deficiency (rice)
• hemoglobin production (pigs)
• healthier fats (pigs)

Question 9

concerns of GMOs

Answer 9

• transfer of genes to other species
• out compete native crops
• long-term effects on human health are unknown
• ownership problems, cost to individual farmers
• fields with round-up resistant crops are sprayed with chemicals

Question 10

regulation of GMOs

Answer 10

International
> agreement between 130 countries
Within the U.S.
> FDA
> EPA
> USDA
> NIH

Question 11

how are GMOs created?

Answer 11

• extract DNA
• amplify specific DNA sequence using PCR
• use gel electrophoresis, restriction enzymes, and ligase to place the DNA segment into a plasmid
• transform E.coli
• select for transformed cells and confirm with a restriction digest
• move to an expression vector
• transform the organism

Question 12

Thermus aquaticus (Taq)

Answer 12

• bacteria isolated from hot springs in Yellowstone in 1966
• extreme thermophile
• optimal growth at 70-75 degrees C
• Taq DNA polymerase was isolated and later commercialized in 1980s

Question 13

First Step: Denature DNA (95 C)

Answer 13

• separates two strands

Question 14

2nd Step: Anneal Primers (55-65 C)

Answer 14

• allows primers to pair with their complementary sequences on the DNA sample

Question 15

restriction enzymes

Answer 15

• recognizes a specific DNA sequence at which it cuts both strands of the sugar- phosphate backbone of DNA
• first identified in bacterial cells, where they function as protection against viral infection

Question 16

restriction-modification systems

Answer 16

• methylate the restriction enzyme recognition sequences in bacteria, to protect the bacterial sequences from digestion

Question 17

features of restriction enzymes (part 1)

Answer 17

• common in bacteria and are named after the species in which they were identified
• single-stranded segments produced by some restriction enzymes cuts are called STICKY ENDS and can base-pair with complementary sequences
• 2 DNA molecules with complementary sticky ends can thus be combined by complementary base pairing

Question 18

part 2

Answer 18

• EcoRI recognizes the palindromic sequence
• then cuts to create “sticky ends” with single-stranded segments at the ends of each fragment

Question 19

part 3

Answer 19

• some RE leave blunt ends, with no single-stranded overhangs
• the sequences recognized by RE vary from 4 to 8 bp
• the number & size of fragments produced by digesting with a given restriction enzyme depends on the size of the genome and the relative abundance of each nucleotide

Question 20

restriction maps

Answer 20

provide info for further experiments, such as subcloning, cutting of DNA into smaller fragments in order to separately clone subsections of the sequence
- restriction digests with more than one enzyme, including double digests, help generate accurate maps

Question 21

digestion of a large genome

Answer 21

• digesting the DNA of a large genome with a restriction enzyme yields a smear of many fragments of differing sizes
  Ex: human genome digested with EcoRI, produces 730,000 diff pieces