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Flashcards in Recombinant DNA and Biotechnology Deck (18)
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1

Recombinant DNA

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

2

DNA cloning

technique that can produce large amounts of a desired sequence

3

two reasons why bacteria can be made is..

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)

4

Vectors

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

5

Restriction enzymes (restriction endonucleases)

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

6

Some restriction enzymes produce offset cutes, yielding

sticky ends of the fragments

7

sticky ends

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

8

DNA cloning can be used to produce

DNA libraries

9

DNA libraries

large collections of known DNA sequences
these sequences could equate to the genome of an organism

10

Genomic libraries

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

11

cDNA (complementary DNA)

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.

12

Hybridization

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

13

Polymerase Chain Reaction (PCR)

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)

14

Gel Electrophoresis

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

15

Southern blot

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.

16

Gene Therapy

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

17

Transgenic mice

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

18

Knockout mice

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