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Molecular Genetic Analysis and Biotechnology

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Gaia Bistulfi

on 8 April 2015

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Transcript of Molecular Genetic Analysis and Biotechnology

MOLECULAR GENETIC ANALYSIS
and BIOTECHNOLOGY

BIO302-L
bistulfi@dyc.edu
Molecular Genetics
...includes the cellular processes
of REPLICATION, TRANSCRIPTION,
and TRANSLATION.
DNA is in the nucleus of the cell
DNA
DNA is COMPLEMENTARY
and ANTI-PARALLEL
The central DOGMA of
Molecular Biology
The new "extended" dogma:
Recombinant DNA technology
(Genetic engineering)
...locates, isolates, alters and studies DNA segments.
"Recombinant" because often combines DNA from different organisms.
Biotechnology
...refers to the industry that uses
recombinant DNA technology to make
new products (drugs, diagnostic kits,
agriculture.......)
Let's make some human
Growth Hormone!
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
ENDONUCLEASES
Cut double strand DNA (both strands) at specific sequences
Restriction enzymes
Restriction enzymes:
sticky or blunt ends?
Restriction enzymes
cohesive (sticky) ends
Viewing DNA fragments
Gel electrophoresis
Southern, Northern, Western Blotting...
DNA
RNA
Proteins
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
Cloning vector:
A stable, replicating DNA molecule to which a foreign DNA fragment can be attached.
IT NEEDS
We cloned our gene! And now what?
TRANSFORMATION!
Inserting the vector (or PLASMID)
into bacteria cells
Selection
Expression vectors
9
6-Extract the protein (hGH), from bacteria (only if no post-translational modifications).
Great! I
think
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?
CLONING!
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.
DNA polymorphisms
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
POLYMORPHISM analysis
Of interest e.g. if associated with a specific phenotype!
WHY?
The whole human genome was "sequenced"! (2000)
HOW?
DNA sequencing:
the dideoxy method
Each sample is set up in 4 reactions with DNA pol
Each reaction contains 4dNTPs and ONE ddNTP
DNA sequencing:
the dideoxy method
What would happen in each tube?
Load the 4 reaction on a gel!
SUMMARIZING...
The dogma: DNA replication, transcription, translation
Restriction enzymes
Gel electrophoresis
PCR
Cloning (plasmids)
RFLP
DNA sequencing
QUESTIONS?
EX 16/19
Ex 20
EX 24
Full transcript