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Molecular Genetic Analysis and Biotechnology
Transcript of Molecular Genetic Analysis and Biotechnology
...includes the cellular processes
of REPLICATION, TRANSCRIPTION,
DNA is in the nucleus of the cell
DNA is COMPLEMENTARY
The central DOGMA of
The new "extended" dogma:
Recombinant DNA technology
...locates, isolates, alters and studies DNA segments.
"Recombinant" because often combines DNA from different organisms.
...refers to the industry that uses
recombinant DNA technology to make
new products (drugs, diagnostic kits,
Let's make some human
1- Find it in 3.2 x10 bp of DNA...
2-Separate it from the rest of the DNA...
3- Put it in a bacterial cell, in a stable form! AND it should be replicated...
4-It should be properly transcribed (and translated!)
5- Find out which bacteria cells carry our gene...
Cutting and joining DNA fragments
RESTRICTION ENZYMES or
Cut double strand DNA (both strands) at specific sequences
sticky or blunt ends?
cohesive (sticky) ends
Viewing DNA fragments
Southern, Northern, Western Blotting...
Many recombinant DNA methods require many copies of a specific DNA fragment
Polymerase Chain Reaction (PCR)
Uses "DNA polymerase" to make many copies of one fragment of DNA
A stable, replicating DNA molecule to which a foreign DNA fragment can be attached.
We cloned our gene! And now what?
Inserting the vector (or PLASMID)
into bacteria cells
6-Extract the protein (hGH), from bacteria (only if no post-translational modifications).
I "cut" my DNA. How do I "see" it? How do I separate the fragments?
"Finding" a fragment
For example...even if you could cut out from the genome only your gene of interest...YOU WOULD NEVER BE ABLE TO SEE IT ON A GEL! Many copies are needed!
GREAT! But What if I want to separate a gene from organism A to put it in organism B?
What are DNA "POLYMORPHISMS"?
Individuals of a same species can reproduce, because they have the same genes in the same location of the genome.
What determines a "species"?
What makes us different from each other then?
Genes come in different ALLELES.
POLYMORPHISMS are variations of a DNA sequence.
Not all polymorphisms give rise to a different allele.
Some may change susceptibility to restriction enzymes!
What would happen if I cut these 2 samples of DNA with HaeIII?
RFLP= RESTRICTION FRAGMENT LENGHT
Of interest e.g. if associated with a specific phenotype!
The whole human genome was "sequenced"! (2000)
the dideoxy method
Each sample is set up in 4 reactions with DNA pol
Each reaction contains 4dNTPs and ONE ddNTP
the dideoxy method
What would happen in each tube?
Load the 4 reaction on a gel!
The dogma: DNA replication, transcription, translation