Restriction Enzymes (endonucleases)

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2 In order to understand and eventually manipulate DNA (human or otherwise) an array of DNA technologies have been developed. Here are some of the tools: Restriction Enzymes (endonucleases) In order to manipulate DNA, Bioengineers have developed a series of bacterial enzymes that can be used to cut and paste selected fragments of DNA Goal is to create a new engineered sequence of DNA called Recombinant DNA How it Works: (i) A Plasmid is extracted from a prokaryotic cell (usually bacteria) and Donor DNA is extracted from a eukaryotic cell

3 (ii) Plasmid and Donor DNA are treated with the same Restriction Enzyme The Restriction Enzyme will cut the Plasmid and Donor DNA in the same specific sites creating Restriction Fragments with single stranded ends called sticky ends

4 (iii) The Donor gene sequence (restriction fragment from the donor DNA) is then spliced into the plasmid by matching the sticky ends DNA Ligase is introduced to seal the sticky ends and make the splicing permanent (iv) Recombinant Plasmid is introduced into a new bacteria cell Spliced DNA (inserted gene) is replicated along with the original host bacterial DNA (v) Products from the inserted eukaryotic gene are isolated and collected in large amounts OR Recombinant bacteria is isolated for use elsewhere

5 Methods of Cloning DNA Fragments (DNA Amplification) 1. Bacterial Cloning with Plasmids Once a DNA fragment has been isolated it is necessary to copy or clone it in order to further analyze and manipulate it Cloning Vector a molecule that replicates foreign DNA within a cell and can be moved into and out of a host cell

6 How it Works: (i) A target sample of DNA is broken into a specific pattern of restriction fragments by a restriction enzyme (or enzymes) (ii) Restriction fragments are spliced into a bacterial plasmid (using restriction enzymes and DNA Ligase) creating a recombinant plasmid (iii) The recombinant plasmid is returned to the bacterial cell. As the cell multiplies (reproduces) is replicates the recombinant plasmid containing foreign DNA This allows millions of copies of the DNA to be made Problems: The process is very slow To process is very difficult for small DNA fragments

7 2. Polymerase Chain Reaction (PCR) Developed in 1986 the CSI Method Used to target and amplify small specific DNA sequences How It Works: (i) (ii) DNA fragment is isolated (by restriction enzyme) DNA fragment is placed into a solution with nucleotides and primers (iii) Solution is heated to break the hydrogen bonds between the 2 DNA strands and open the double helix (strands are separated) (iv) Heat resistant DNA Polymerases (I and III) are added to replicate the DNA (v) In just over ONE MINUTE both DNA strands are replicated (vi) The solution is heated again and the cycle repeats itself. Each cycle double the amount of DNA PCR can quickly generate billions of copies of a DNA sequence for analysis

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9 Methods of DNA Sequencing and Identification There are a number of techniques used to identify and sequence DNA fragments Many of these techniques were pioneered by an English Biochemist named Frederick Sanger Sanger is the only person to have been awarded the Nobel Prize twice in Chemistry. Received his second Nobel Prize in 1980 for a method of sequencing DNA that is now called the Sanger method

10 Method #1: Gel Electrophoresis Developed by Dr. Frederick Sanger Used to separate DNA fragments according to their mass and charge Enables DNA fragments to be separated so they can be analyzed DNA Fingerprinting How it works: (i) (ii) Restriction enzymes are added to a sample of DNA to cut the DNA into fragments A semi-permeable gel is formed with small wells at one end Small amounts of the sample DNA are placed in the wells (iii) The gel is placed in a solution and an electrical field is set up so that one end of the gel is positive and the other end is negative (iv) The ( ) charged DNA fragments travel towards the (+) end of the gel. The smaller the fragment is, the faster the fragment moves through the gel and the further away it moves from the well.

11 For Fingerprinting Purposes: With the exception of identical twins, no two individuals have the same fragment pattern so, if a DNA sample was taken from a crime scene (and then amplified by PCR), authorities could test to see if the gel pattern produced from a DNA sample matches the gel pattern from a suspect Paternity Tests are based on this technology. If the gel pattern of a child and mother are known, the gel pattern of the father can be determined and then compared to possible fathers in a test

12 Method #2 RFLP Analysis Restriction Fragment Length Polymorphism Analysis is a process used to learn the nucleotide sequence of alleles, chromosomes and eventually entire genomes How it Works: (i) (ii) DNA samples to be `sequenced` are added to specific restriction enzymes which create restriction fragments The restriction fragments are run through gel electrophoresis. Each sample creates a characteristic pattern of bands (iii) The DNA fragments on the gel are treated with a chemical which `denatures`them (makes them single stranded) and the single strands are `blotted`onto a special filter paper bands are transferred to paper (iv) Radioactive Probes (radioactively tagged nucleotides) are added to the bands on the filter paper. The probes attach to complimentary bases on the single stranded fragments (v) A sheet of photographic film is laid over the `blot`. The radioactivity creates an image identifying the probe sequence which is used to determine the DNA sequence

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14 Method #3 Chain Termination Sequencing Aka The Sanger Method This method pioneered by Dr. Frederick Sanger is used to sequence entire genomes How it Works: (i) (ii) DNA is broken into small fragments by restriction enzymes The nucleotide at the end of each fragment is tagged with a fluorescent marker (iii) The fragments are run through gel electrophoresis The resulting fingerprint shows which fragment ends with which nucleotide (ex) if fragments were tagged with G markers and the end of a 10 nucleotide fragment was successfully tagged then the 10 th nucleotide is a G (iv) All of the results are fed through a computer and a sequence is generated

15 The Human Genome Project In the late 1990 s, computer technology advanced far enough to sample and analyze large amounts of DNA In 2001, the first map of the entire human genome is mapped Consists of 3 billion base pairs (6 billion nucleotides) genes (suggesting that genes may have three or more functions) Learned location of genes Learned that humans are 99.9% identical Only 1 in 1000 nucleotides are different

16 If we know the function and location of a gene should we be able to change the sequence? Can a gene sequence be copyrighted or trademarked? Should employers be able to ask for a genetic profile?