6.5 Recombinant DNA and Biotechnology [HY]

Question 1

What is required in cloning?

Answer 1

• Requires that the investigator ligate the DNA of interest into a vector, to form a recombinant vector

Question 2

Vectors

Answer 2

• usually bacterial or viral plasmids that can be transferred to a host bacterium after insertion of the DNA of interest
• The bacteria are then grown in colonies, and a colony containing the recombinant vector is isolated
• bacteria can then be made to express the gene of interest (generating large quantities of recombinant protein), or can be lysed to reisolate the replicated recombinant vectors (which can be processed by restriction enzymes to release the cloned DNA from the vector).
• requires an origin of replication and at least one gene for antibiotic resistance to allow for selection of colonies with recombinant plasmids
• DNA vectors contain at least one sequence, if not many, recognized by restriction enzymes

Question 3

Restriction enzymes (restriction endonucleases)

Answer 3

• enzymes that recognize specific double-stranded DNA sequences. These sequences are palindromic, meaning that the 5′ to 3′ sequence of one strand is identical to the 5′ to 3′ sequence of the other strand (in antiparallel orientation)
• In bacteria, they act as part of a restriction and modification system that protects the bacteria from infection by DNA viruses
• Some restriction enzymes produce offset cuts, yielding sticky ends on the fragments (Sticky ends are advantageous in facilitating the recombination of a restriction fragment with the vector DNA.

Question 4

What happens if the vector is cut with the same restriction enzyme?

Answer 4

• It allows the fragments to be inserted directly into the vector

Question 5

DNA libraries

Answer 5

• large collections of known DNA sequences; in sum, these sequences could equate
  to the genome of an organism

Question 6

Genomic libraries

Answer 6

• contain large fragments of DNA, and include both
  coding (exon) and noncoding (intron) regions of the genome
• Restriction endonuclease DNA ligase to make library
• Contains nonexpressed sequences of chromosomes
• Contains promoter and enhancer sequences.
• Gene can not be expressed in cloning host (recombinant proteins)
• Can not be used for gene therapy or constructing transgenic animals

Question 7

cDNA (complementary DNA) Libraries

Answer 7

• libraries are constructed by reverse-transcribing processed mRNA
• lacks noncoding regions, such as introns, and only includes the genes that are expressed in the tissue from which the mRNA was isolated.
• only cDNA libraries can be used to reliably sequence specific genes and identify disease-causing mutations, produce recombinant proteins (such as insulin, clotting factors, or vaccines), or produce transgenic animals.
• No promoter or enhancer sequences

Question 8

Expression libraries

Answer 8

• another name for cDNA libraries

Question 9

Polymerase chain reaction (PCR)

Answer 9

• an automated process that can produce millions of copies of a DNA sequence without amplifying the DNA in bacteria.
• requires primers that are complementary to the DNA that flanks the region of interest, nucleotides (dATP, dTTP, dCTP, and dGTP), and DNA polymerase. The primer has high GC content (40–60% is
  optimal), as the additional hydrogen bonds between G and C confer stability
• During PCR, the DNA of interest is denatured, replicated, and then cooled to allow reannealing of the daughter strands with the parent strands

Question 10

Gel Electrophoresis

Answer 10

• technique used to separate macromolecules, such as
  DNA and proteins, by size and charge.
• DNA goes to anode
• the longer the DNA strand, the slower it will migrate in the gel
• preferred gel for DNA electrophoresis is agarose gel

Question 11

Southern Blotting

Answer 11

• used to detect the presence and quantity of various DNA strands in a sample
• DNA is cut by restriction enzymes and then separated
  by gel electrophoresis.
• The DNA fragments are then carefully transferred to a membrane, retaining their separation
• The membrane is then probed with many copies of a single-stranded DNA sequence.
• The probe will bind to its complementary sequence and form double-stranded DNA.

Question 12

DNA Sequencing Mech.

Answer 12

• Template DNA + Primers + Polymerase + AT- GC + Dideoxyribonucleotide (has a modded base so can’t extend chain) -> Creates sample with lots of fragments -> Fragments separated by size -> Bases read in order

Question 13

Gene Therapy

Answer 13

• intended for diseases in which a given gene is mutated or inactive, giving rise to pathology
• By transferring a normal copy
  of the gene into the affected tissues, the pathology should be fixed, essentially curing the individual.
• most gene delivery vectors in use are modified viruses
• Randomly integrated DNA poses a risk of integrating near and activating a host oncogene

Question 14

Transgenic mice

Answer 14

• altered at their germ line by introducing a cloned gene into fertilized ova or into embryonic stem cells

Question 15

Transgene

Answer 15

• The cloned gene that is introduced
• If the transgene is a disease producing allele, the transgenic mice can be used to study the disease process from early embryonic development through adulthood.
• the transgene will also be passed to their offspring.
  The transgene coexists in the animals with their own copies of the gene, which have not been deleted.
• transgenic mice may each contain a different number of copies of the transgene

Question 16

Chimera

Answer 16

transgenic cells and host blastocyst