(From http://generalhorticulture.tamu.edu) |
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Analogy |
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DEFINITIONS Base Pairing of
Nucleic Acids between the double strands of DNA gene - a length of
DNA that codes for the production of a protein or protein subunit. |
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The
Central Dogma of Molecular Biology
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Gene - A
More Detailed Description (From: http://www.accessexcellence.org)
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Steps
Leading from Gene to Protein (From: http://www.accessexcellence.org)
Legend: The final
level of a protein in the cell depends on the efficiency of each step and on
the rates of degradation of the RNA and protein molecules. (A) In eucaryotic
cells, the initial RNA molecule produced by transcription (the primary
transcript) contains both intron and exon sequences. Its two ends are
modified, and the introns are removed by an enzymatically catalyzed RNA
splicing reaction. The resulting mRNA is then transported from the nucleus to
the cytoplasm, where it is translated into protein. Although these steps are
depicted as occurring one at a time, in a sequence, in reality they often
occur simultaneously. For example, the RNA cap is typically added and
splicing typically begins before the primary transcript has been completed.
(B) In procaryotes, the production of mRNA molecules is simpler. The 5´ end
of an mRNA molecule is produced by the initiation of transcription by RNA
polymerase, and the 3´ end is produced by the termination of transcription.
Since procaryotic cells lack a nucleus, transcription and translation take
place in a common compartment. In fact, translation of a bacterial mRNA often
begins before its synthesis has been completed.
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Restriction Enzyme Action of EcoRI
(From: http://www.accessexcellence.org)
The EcoRI restriction enzyme--the first restriction enzyme isolated from E. Coli bacteria--is able to recognize the base sequence 5' GAATTC 3'. Restriction enzymes cut each strand of DNA between the G and the A in this sequence. This leaves "sticky ends" or single stranded overhangs of DNA. Each single stranded overhang has the sequence 5" AATT 3'. These overhanging ends will bond to a fragment of DNA which has the complementary sequence of bases. See text of Background Paper for additional details. |
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(From: http://www.accessexcellence.org)
Process by
which a plasmid is used to import recombinant DNA into a host cell for
cloning.The plasmid carrying genes for antibiotic resistance, and a DNA
strand, which contains the gene of interest, are both cut with the same restriction
endonuclease. They have complementary "sticky ends." The opened
plasmid and the freed gene are mixed with DNA ligase, which reforms the
two pieces as recombinant DNA. This produces recombinant DNA.This recombinant DNA
stew transforms a bacterial culture, which is then exposed to antibiotics.
All the cells except those which have been encoded by the plasmid DNA
recombinant are killed, leaving a cell culture containing the desired
recombinant DNA. DNA cloning allows a copy of any specific part of a DNA (or
RNA) sequence to be selected among many others and produced in an unlimited
amount. This technique is the first stage of most of the genetic engineering experiments:
production of DNA libraries, PCR, DNA sequencing, et al.
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Restriction Enzymes Identify Short DNA Sequences
( from http://web.onetel.net.uk/~jbwhammond/REnz1.htm
A restriction map shows the positions at which specific short base sequences (i.e. restriction enzyme recognition sites) occur in a DNA molecule. Restriction maps are made using a specific type of endonuclease (an enzyme which cuts DNA within the molecule NOT at the ends!).
All cells contain a range of nucleases, most have fairly broad preferences as to where they will attack nucleic acids. Bacterial cells produce a special type of endonuclease, called restriction endonucleases (REs), which cut double-stranded DNA only at specific, short base sequences.
REs protect bacterial cells from attack by bacteriophages. Phage DNA entering the cell is disabled by RE attack. The cell's own DNA is protected by addition of a methyl group to one of the bases at the site where the endonuclease would cut it. Methylation is carried out by an enzyme called a methylase which matches with the RE present in the cell.
There are three types of RE (Types I, II and III). They are grouped according to their mechanism of action.
Type II REs, more commonly known as
restriction enzymes, are the most researched and are used widely in DNA
manipulation and analysis. They recognise DNA sequences from 4-16 bp long,
depending on the enzyme, and cut between specific bases within this sequence.
(The sequence is called the recognition site). The recognised sequence and site
of cut is shown for two commonly used restriction enzymes, EcoR1 and Not 1 are
shown below.
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Two examples of restriction enzyme target sites. The arrows show the positions at which the two DNA strands are cut, the red line shows that for these enzymes, the cut leaves a short section of single-stranded DNA at the end of each cut fragment.
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Details of many more restriction enzymes can be found at the www ReBase site and in molecular biology suppliers catalogues and websites.
Using restriction enzymes in mapping
When DNA from the same source is digested with a particular restriction enzyme it will always give a set of the same sized fragments. For example if lambda bacteriophage DNA is cut with EcoR1 we know that it will give six fragments of the sizes: 21.23, 7.42, 5.8, 5.65, 4.87, 3.53 kbp. This is because, mutations apart, the phage sequence will always be the same, and so EcoR1 cutting sites will always be present in the same places. The fragments can be separated and their sizes determined by agarose gel electrophoresis.
We can use the positions of restriction enzyme sites as convenient markers along DNA sequences. The map obtained can be used for DNA identification and to plan DNA manipulations.
Finger Printing
Gel
Showing Banding from use of Different Restriction Enzymes
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Southern Blotting: Gel Transfer(From: http://www.accessexcellence.org)
Detection of
specific DNA fragments by gel-transfer hybridization (Southern blotting). (A)
The mixture of double-stranded DNA fragments generated by restriction
nuclease treatment of DNA is separated according to length by
electrophoresis. (B) A sheet of either nitrocellulose paper or nylon paper is
laid over the gel, and the separated DNA fragments are transferred to the
sheet by blotting. The gel is supported on a layer of sponge in a bath of
alkali solution, and the buffer is sucked through the gel and the
nitrocellulose paper by paper towels stacked on top of the nitrocellulose. As
the buffer is sucked through, it denatures the DNA and transfers the
single-stranded fragments from the gel to the surface of the nitrocellulose
sheet, where they adhere firmly. This transfer is necessary to keep the DNA
firmly in place while the hybridization procedure (D) is carrried out. (C)
The nitrocellulose sheet is carefully peeled off the gel. (D) The sheet
containing the bound single-stranded DNA fragments is placed in a sealed
plastic bag together with buffer containing a radioactively labeled DNA probe
specific for the required DNA sequence. The sheet is exposed for a prolonged
period to the probe under conditions favoring hybridization. (E) The sheet is
removed from the bag and washed thoroughly, so that only probe molecules that
have hybridized to the DNA on the paper remain attached. After
autoradiography, the DNA that has hybridized to the labeled probe will show
up as bands on the autoradiograph. An adaptation of this technique to detect
specific sequences in RNA is called Northern blotting. In this case mRNA
molecules are electrophoresed through the gel and the probe is usually a
single-stranded DNA molecule.
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