1. Human awareness

2.  M16.1 Know that the DNA can be extracted from cells  Genetic engineering and /or genetic modification have been made possible by isolating and manipulating genes extracted from the DNA of species.  Genetic engineering means taking genes from one organism and putting them into another to create a modified gene or altering them and putting them back into the original organism in an altered state.

3.  Other uses of genetic engineering are:  Producing other proteins like vaccines  Improving plants in terms of pest resistant crops  Improving animals like better wool, more milk yield, improved meat etc

4.  Locating a gene of interest on the chromosome  Using enzymes to isolate  Sequencing the gene to determine the structure  Inserting the gene into cells of the target organism  Growing the transformed cells into a complete organism

5. Restriction enzymes are the tools used to cut DNA in various places. Found in bacteria Different species=different enzymes Some work at boiling point of water 1000 types known

6.  IDENTIFYING AND ISOLATING THE RIGHT GENE:  Once DNA is extracted and isolated from nuclear proteins  restriction enzymes cut DNA into smaller fragments ( these enzymes are found in bacteria)  to isolate the specific gene in question two methods are used:

7.  must know a short sequence of nucleotides in a gene  if you know the protein for which the gene codes, you know the amino acid sequence and should be able to deduce the nucleotides  a short segment of a single strand of DNA or RNA with a sequence of bases that is complimentary to part of the required genes is selected  this is radioactively labelled and mixed with the double stranded DNA fragments that contain the gene  the solution is heated and separated  on cooling, some of the probe molecules will bind to the DNA fragments containing the specific complimentary base sequence and identifies and locates the gene  DNA ligase that you remember helps seal the breaks in DNA during replication, is used to form the recombinant DNA  By inserting the DNA into a host organism it is possible to make multiple copies of the required gene- this is then the cloning process addressed next  See page 44 Essentials text

8.  Cloning Genes:  to clone genes that have already been identified and isolated, involves bacteria  as stated, bacteria have little circles of DNA called plasmids  these are separated from the bacteria Chromosomes

9.  plasmids can be removed and cut with special restriction enzymes  once cut, you end up with short sticky ended bits of DNA…which can then attach to other strands of DNA …as long as the ends have complimentary nucleotides  This means that biologists can use a certain enzyme to cut the plasmid at a particular point and insert a gene of interest which has been identified in humans and also removed using a probe or antibody method.  The DNA is stuck together using ligase and the plasmids reform into rings

10.  they are mixed with bacterial cells which take them up quite readily  Bacteria reproduces rapidly and each time it reproduces, this particular gene will be copied--- gene cloning  this is a way to produce bio-chemicals like insulin and growth hormone which are made by protein synthesis  It is possible to incorporate “foreign” DNA into other organisms ---this is called transgenic or transgenesis ---dolly the sheep!  this is done so that an animal may be able to produce something we need via their milk etc, or plants by producing a natural insecticide to protect themselves  vaccine production

11.  To introduce the gene to the organism you want to take up the new gene, and therefore altered function, i.e. make a wheat plant be resistant to insects after removing a gene you know does this from say a nettle plant etc you need to :  May use a gene which has been developed using the bacteria plasmid and in terms of plants – a special bacteria called AGROBACTERIUM is used which infects the plant and acts as a natural genetic engineer making the plant take on the new gene see page 46

12.  In animals there are three steps:  Isolate the gene-using the restriction enzymes  Inject the recombinant DNA with a fine glass needle into the pro-nuclei in an embryo of that organism 12-14 hours after fertilization  Transfer the embryo to a recipient mother where it can grow taking up that gene into all its cells as in normal division  These are the transgenic organisms diagram page 47

13.   READ PAGE 48 OF Essentials and chapter 6?

14.  M17.1 Understand that segments of DNA can be multiplied and then have their base sequence identified.  Humans can sequence even small amounts of DNA:  1960-Fred Sanger  heated DNA so it separated  added DNA polymerase and excess of the base nucleotides A,C,G, and T  Normally the primers would stick to the single strands and polymerase would join the free nucleotides with the corresponding bases until the double helix was finished

15.  Using four different test tubes he was able to see when the sequence was stopped by identifying which was the last nucleotide added before it couldn’t add his altered A or T etc.. this meant he could determine the sequence of DNA  DNA can be copied and amplified or copied lots to the point where there is lots to analyse  This means you need to know the start of the sequence of DNA you want to copy eg ATG and use a primer which is a short piece of DNA that matches this section you want to copy- i.e. TAC, the primer joins to one strand of the DNA and prevents it re-joining as a double helix

16.  special DNA polymerase enzymes from bacteria that don’t denature with heat along with lots of copies of the primers and free nucleotides are put in with DNA to increase the chance of the primer finishing the strand of DNA and not allowing it to re-join into its original double helix; but to produce two copies of the double helix  repeated heating and cooling (as long as there is lots of primer and free nucleotides) lets us make lots of copies of the DNA  THE POLYMERASE CHAIN REACTION –read page 50  EXPLORE DNA fingerprinting page 51 17.2