જ⁀➴ recombinant dna technology

what is recombinant DNA technology?

process of combining DNA from different sources to create new genetic combinations

Examples of recombinant DNA techniques

1. cloning

2. DNA libraries

3. Expression vectors

4. PCR

5. Gene therapy

what is cloning?

the production of an exact copy of a DNA segment

what are cloning vectors?

DNA molecules into which “foreign” DNA is inserted for cloning.

examples of cloning vectors

1. plasmids

2. cosmids

3. bacterial artificial chromosome

4. yeast artificial chromosome

how do cloning vectors work?

1. uses an restriction endonuclease (enzyme) to cut the plasmid and the foreign DNA

2. Then the foreign dna anneals (hydrogen bond together) to the plasmid

3. later is ligated or sealed. the plasmid grows in size and the foreign dna is not visible.

what is a genomic library?

collection of cloned chromosomal DNA fragments representing the entire genome of an organism

how is a genomic library prepared?

1. collect all the DNA (including introns)

2. produce fragnments (cut it)

3. clone the DNa fragments using a vector

what is cDNA?

copies of mRNA molecules

How is cDNA made?

1. start with mRNA from a cell

2. reverse transcriptase will crete the first strand of cDNA

3. ribonuclease will remove the mRNA

4. DNA polymerase will create the second strand of cDNA

5. double stranded cDNA is inserted into a vector, where the bacteria will copy it.

what is RNA expression?

the process of producing RNA from a cDNA or other foreign DNA inserted into aa vector either in vitro (test tube) or a host cell

How is RNA expression achieved?

by placing a promoter sequence (where RNA polymerase binds) next to the inserted DNA in a vector, allowing transcription into RNA

what is protein expression?

vectors are used to synthesize proteins, incuding human protein, in e.coli or other host cells.

how do you express a foreign protein in E.coli?

a cDNA molecule representing the gene of interest is inserted next to a promoter and a ribosome-binding site in the vector.

what is PCR?

a polymerase chain reaction, lab technique to make millions of copies of a specific DNA segment in vitro.

What does PCR include?

1. oglionucleotide primers

2. DNA template

3. deoxynucleotides

4. heat-stable DNA polymerase

What does PCR consist of?

multiple cycles of heating and cooling a reaction mixture 

what are restriction endonuclease?

enzymes that cut DNA at specific sequences 94 to 8 nucleotides long), and create sites for foreign DNA to be inserted.

give an example of a restriction endonuclease.

EcoR1 — recognizes the sequences GAATTC and makes staggered cuts. Cuts between G and A

what type of cut does EcoRV make?

blunt end; non-staggered

what is southern blotting?

DNA fragments are seperated by gel electrophoresis, transfered to. sheet of membrane (blotted), and detected with a DNA probe

what is northern blotting?

RNA molecules such as mRNAs are separated in a gel, transferred to. sheet pf membrane, and detected with a probe.

what is western blotting?

proteins are separated in a gel, transferred to a sheet of membrane, and detected with antibodies. 

what is human gene therapy?

introduction of a functional gene into human cells to cpnnect a defective or mutant gene

what is an expression cassette?

a DNA construct containing a cDNA gene + promotor, used for gene expression in target cells.

how are genes delivered in human gene therapy?

using viral vectors

what are some common viral vectors used in human gene therapy?

1. DNA viruses

2. Retroviruses

What would be an appropriate source of the DNA polymerase used in the PCR procedure to amplify specific DNA segments?

Thermus aquaticus