RECOMBINANT DNA TECHNOLOGY

One of the groundbreaking results of Recombinant DNA

technology was the production of

factor VIII

Missing in patients who have Hemophilia

Factor VIII:

Around the 1980s, Genentech company & Genetics Institute had a race to find how to produce factor VIII artificially thru recombinant DNA technology

 Is the process of using recombinant DNA (rDNA)

technology to alter the genetic makeup of an organism

 The umbrella where recombinant DNA technology is

GENETIC ENGINEERING

Technique that involves the identification, isolation and

insertion of genes of interest into a vector (plasmid or

bacteriophage) to form a recombinant DNA molecule

Genetic engineering

Process of making multiple, identical copies of a particular piece of DNA

GENE / MOLECULAR CLONING

They are genetically modified because of the addition of Green fluorescent gene to produce GFP

Transgenic / genetically modified organisms

is a collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further stud

DNA Library:

Contains representation for all the genes (genome)

Genomic DNA Library

Extracted from existing genomes

Genomic DNA Library

Consists of introns (noncoding) and exons (coding) region

Genomic DNA Library

Constraints representation for the genres that are expressed (mRNA in DNA form)

cDNA Library

(complementary)

Synthesized from the reverse

transcription of different types of

RNA

cDNA Library

(complementary)

Consists of exons only Advantage is that it only contains exons (important region) Before mRNA is released, it will be further processed

cDNA Library

(complementary)

the addition of methyl group by E. coli to the existing bacterial chromosomal DNA

Methylating:

discovered restriction enzymes

Werner Arber (1960s)

It cut in the nucleotide sequence (in the middle).

Proteins that cleave DNA molecules at specific sites, producing discrete fragments of DNA

Restriction Endonucleases:

: cuts in the end of the nucleotide region

Cuts are specific

Restriction Exonucleases

ends of a DNA molecule that end with a

base pair

BLUNT ENDS:

ends of a DNA molecule that and with nucleotide overhang or with extension

 More stable than blunt ends

STICKY ENDS/ COHESIVE/ PROTRUDING ENDS:

produce “blunt” ends

Alul and Haelll

produce “sticky” ends

BamHl, Hindlll and EcoRl

 Molecular glue

 Adds phosphodiester bond

DNA LIGASE

 Example is plasmids

 Agent that can carry a DNA fragment into a host cell in which it is capable of replication

VECTORS

Vectors aka nes

Cloning vehicle

means to cut and paste the genes

Cloning vehicle

Used only for reproducing the DNA fragment

CLONING VECTOR

Comprise origin of replication, unique restriction sites and selectable marker and reporter genes.

CLONING VECTOR

Used for expression of foreign gene to prudence the proteins

EXPRESSION VECTOR

Must have strong promoter and enhancer region (for efficient transcription),

termination codon

EXPRESSION VECTOR