Recombinant DNA and Biotechnology

Question 1

Recombinant DNA

Answer 1

allows a DNA fragments from any source to be multiplied by either gene cloning or polymerase chain reaction (PCR)

Question 2

DNA cloning

Answer 2

technique that can produce large amounts of a desired sequence

Question 3

two reasons why bacteria can be made is..

Answer 3

To express the gene of interest (generating large quantities of recombinant protein) or can be lysed to resolute the replicated recombinant vectors (which can be processed by restriction enzyme to release the cloned DNA from the vector)

Question 4

Vectors

Answer 4

can be transferred to a host bacterium after insertion of the DNA of interest

requires an origin of replication and at lease one gene for antibiotic retsina to allow for selection of colonies with recombinant plasmids

Question 5

Restriction enzymes (restriction endonucleases)

Answer 5

enzymes that recognize specific double - stranded DNA sequences

are isolated from bacteria, which are their natural source

in bacteria, they act as part of a restriction and modification system that protects the bacteria from infection by DNA viruses

Can cut the vector of choice

Question 6

Some restriction enzymes produce offset cutes, yielding

Answer 6

sticky ends of the fragments

Question 7

sticky ends

Answer 7

can facilitate the recombination of a restriction fragment with the vector DNA

Question 8

DNA cloning can be used to produce

Answer 8

DNA libraries

Question 9

DNA libraries

Answer 9

large collections of known DNA sequences

these sequences could equate to the genome of an organism

Question 10

Genomic libraries

Answer 10

contain large fragments of DNA and include in both coding (exon and noncoding (intron) regions of the genome

Question 11

cDNA (complementary DNA)

Answer 11

Libraries that are constructed by reverse - transcribing processed mRNA
Lacks noncoding regions, such as introns and only includes the genes that are expressed in the tissue form which the mRNA was isolated and for this reason, they are sometimes called expression libraries.

Question 12

Hybridization

Answer 12

the joining of complementary base pair sequences
can be DNA - DNA recognition or DNA - RNA recognition
This technique uses two single stranded sequences and is a vital part of polymerase chain reaction and Southern blotting

Question 13

Polymerase Chain Reaction (PCR)

Answer 13

automated process that can produce millions of copies of a DNA sequences without amplifying the DNA in bacteria.
Requires PRIMERS that are complementary to the DNA that flanks the region of interest, nucleotides (A,T,C and G) and DNA polymerase
Needs to be heated to cause the DNA double helix to melt apart (denature)

Question 14

Gel Electrophoresis

Answer 14

a technique used to separate macromolecules, such as DNA and proteins by size and charge

Question 15

Southern blot

Answer 15

used to detect the presence and quantity of various DNA strands in a sample

DNA is cute 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 bid to its complimentary sequence and form double stranded DNA. Probes are labeled with radioisotopes or indicator proteins, both of which can be used to indicate the presence of a desired sequence.

Question 16

Gene Therapy

Answer 16

intended for diseases in which a given gene is mutated or inactive, giving rise to pathology.
Most gene delivery vectors in use are modified viruses

Question 17

Transgenic mice

Answer 17

created by integrating a gene of interest into a germ line or embryonic stem cells of a developing mouse

altered at their germ line by introducing a cloned gene into fertilized ova or into embryonic stem cells. The cloned gene that is introduced is referred to as a TRANSGENE.
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.

organisms that contain cells from two different lineages (such as mice formed by integration of transgenic embryonic stem cells into a normal mouse blastocysts) are called chimeras

Transgenic mice can be mated to select for the transgene

Question 18

Knockout mice

Answer 18

a gene has been intentionally deleted (knocked out).
These mice provide valuable models in which to study human diseases.

created by deleting a gene of interest