April 23rd, 2008

 

Correction:

Sense, Anti-sense.

The strand of DNA that is copied is the template strand (read 3 to 5 direction) and hte mRNA that it yeilds is ("5' to 3')

Exam is Friday.

 

Finish all today Ch 20.

We pick up talking about ...

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Recombinant DNA Technology.

Hosts:

 

Bacteria

Most typical = E.coli

Advantages - easy to grow (intestines) in culture, and easy to isolate plasmid DNA.

Disadvantage - no splicing of mRNA

// as soon as its transcribed its immeadiately translated -- and if its

So Eukaryotic genes that have introns cannot be expressed (protein) in bacteria.

 

Phages (Bacterial viruses),Yeast

 

Methods of Genetic Engineering

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*Look at cloning slide LE 20-3

Sticky ends are complementary to one another.
Advantage of Rea __ ENDS are defined - particular DNA sequence, so when you digest it with enzyme, you can see

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Methods of Genetic Engineering

 

1.) Gene Cloning - Isolate one gene of interest from an organism. (tens of thousands of genes, its like a needle in a haystack!)

a.) make a gene library = a collection of all the genes in an organism.

There are two types of Libraries:

• Genomic Libraries - contains ALL the DNA of the organism (-including repetitive sequences, introns of genes, the genes themseles of course, regulatory sequences, everything!)

 

• cDNA Libraries - a cDNA copy of all the mRNA in a cell.

  //we can convert the mRNA with reverse transcriptase --> cDNA - and  with DNA polymerase---> Double Stranded DNA

Contains only Genes that are expressed in that cell.

~ 1% of the genome

 

mRNA - mature mRNA -> so there are NO introns.

These proteins can be expresed correctly by bacteria

 

Another advantage: Each cell type has a different mRNA profile - which genes are expressed.

(b) identify the gene of interest.

1- use a DNA probe = DNA sequence that is used to identify your gene -

(may be of a different species etc)  Sequence will be complementary to your gene of interest

Labeled with some type of marker - usually a radioactive probe is used.

 

1)

Southern Blotting -

See Slide LE-20-5

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Grow matrials up - dilute them so single colonies on plate,

break cells and put on filter

Radioactive proobe = melt the DNA - break hydrogen onds - single strand

Single Cell DNA wash over filter - if finds complementary, then it binds to it,

Expose it to a film, and then you can Identify which bacteria contain the gene you were interest in

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2 - Antibody probe - works on a cDNA protein expression library

Bacteria--->protein--> some of the bacteria (only those that are making the protein of interest) are labeled with the antibody probe (labeled - raioactiity, usualy enzyme)

 

Probes are always labeled.

 

Electrophoresis - method to separate DNA or proteins based on size.

(because restriction pattern is different, vics blood on defendent)

 

PCR = Polymerase chain reaction - method to make millions of copies of a specific DNA target sequence

used for Ancient DNA like mammoths and mummies etc where things are degraded and almost gone. 

 

Know basic way things work:

LE 20-7

A special type of DNA polymerase that works in high temperatures to melt the DNA

Denature it - break H bonds

Primers that bind to sequence

DNA polymerase elongates the sequence.

 

DNA Sequencing:

Human Genome Project -

 

Linkage mapping - genetic technique

genes on the chromosomes in order.

 

• DNA is isolated and make Fragments -- Overlapping.
• To tell if they're overlapping or not make Rest EnzymeDigest

-------      -----

------      ------      ------ etc.

Then Sequencing and assembled the sequence.

 

Good for things without rpetitive sequences. - you've no idea for order.

Sequencing things that don't have an order like stuff from the ocean.

 

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NEW method for sequencing DNA 454 pyrosequencing -- on

Original -- NEAT SLIDES

James Watson - less than 1/5 million dollars.

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Microarrays - comparing gene expression in different cell types

 

//G