1. Chapter 20: DNA Technology

2. Important Terminology:  Recombinant DNA: DNA in which nucleotide sequences from 2 different sources (can be from different species) are combined into the same DNA molecule.  Genetic engineering: The direct manipulation of genes for practical purposes (such as producing needed proteins) – often relies on making recombinant DNA.  Biotechnology: The manipulation of organisms or their components to make useful products (modify genes and move them between organisms giving the organisms the ability to make a new protein).  In vitro: refers to procedures conducted OUTSIDE of a living organism.

3. Gene Cloning: preparing gene-sized pieces of DNA in multiple identical copies.  Uses:  To make many copies of a gene (could then be transferred to other organisms).  To produce a protein product.

4.  Made possible by discovery of restriction enzymes.  These are naturally occurring enzymes in bacteria that cut up foreign DNA.  Very specific – each restriction enzyme only “recognizes” a particular DNA sequence – a.k.a. restriction site - and cuts both strands at specific points within this restriction site.

5.  Therefore, copies of DNA molecules always yield the same restriction fragments.  Restriction fragments are double-stranded DNA fragments. Because of the action of restriction enzymes, many will have at least one single- stranded end – called a sticky end.

7.  These will bond with complementary stretches on other DNA molecules that were cut with the same restriction enzyme. Use DNA ligase to make bond permanent.  See: http://highered.mcgraw- hill.com/olc/dl/120078/bio37.swf http://highered.mcgraw- hill.com/olc/dl/120078/bio37.swfhttp://highered.mcgraw- hill.com/olc/dl/120078/bio37.swf  In this way, we make recombinant DNA: DNA that has been spliced together from 2 different sources.

9. Finally! Cloning a Eukaryotic Gene in a Bacterial Plasmid The following is general procedure for using bacteria to make many copies of a gene of interest…  The plasmid is our cloning vector: carrying foreign DNA into a cell and replicating there.  The following is ONE example of a cloning procedure. There are many variation but notice that the plasmids used contain genes that will make the identification of bacteria containing recombinant plasmids easy.

11.  The purpose of using a plasmid with ampicillin resistance: only bacteria who have taken up a plasmid will be able to grow on medium containing ampicillin. So – you will know that those bacteria have been transformed.

12.  Purpose of the lacZ gene: this gene encodes for an enzyme that breaks down lactose & another sugar called X- gal. When it breaks down X-gal, it makes a blue byproduct. If this gene is disrupted, it will not function and won’t break down the X-gal. So, plasmids that have taken up foreign DNA will form white colonies because this gene is disrupted, plasmids that have not, will form blue colonies.

13. http://highered.mcgraw-hill.com/olc/dl/120078/micro10.swf http://bcs.whfreeman.com/stryer/cat_040/ch06/ch06xd06.htm http://www.phschool.com/science/biology_place/labbench/lab6/intro.html

14.  At the end of this procedure, we will have many colonies of bacteria containing many different human DNA fragments. Now – we must identify the colony that has our gene of interest – the one that we want to make many copies of.  To do this, you can look for the gene itself or its protein product.

15.  A process called nucleic acid hybridization is used to look for gene:  If part of the nucleotide sequence is known (maybe by working backward from its protein), a complementary piece of DNA can be made that will bind to it. This is called a nucleic acid probe.  The probe can be labeled with a radioactive isotope or fluorescent molecule and then identified.

16.  Once you know which colony has your gene of interest, you can isolate it and allow the bacteria to replicate – this will produce many bacterial clones – all of which have copies of the desired human gene.

18.  What is the practical application of this procedure?  Can grow large colonies of bacteria containing gene. This gene could be used for other procedures – as a probe to identify similar genes in other organisms.  Also, if the bacteria will express the inserted gene, we can harvest the desired protein product (we do this to obtain human growth hormone, insulin, etc).

19.  The entire procedure above results in MANY plasmids containing different fragments of the original genome. These are often saved and referred to as a genomic library. (Can use them to look for other genes of interest).  Read about making cDNA from mRNA present in cells! You are responsible for this information!

20.  Polymerase Chain Reaction: method by which any piece of DNA can be quickly amplified (copied many times) without being inside a cell.  PCR allows any sequence of DNA to copied many times – in vitro. The use of specific primers can also allow you to make copies of a specific sequence.

21.  PCR (which is now usually done in a machine) involves a cycle of reactions that can repeat over and over to copy DNA. See: http://highered.mcgraw- hill.com/olc/dl/120078/micro15.swf http://highered.mcgraw- hill.com/olc/dl/120078/micro15.swfhttp://highered.mcgraw- hill.com/olc/dl/120078/micro15.swf

24.  Restriction Fragment Analysis  Use gel electrophoresis to sort DNA fragments (resulting from treatment with a restriction enzyme) by size.

25.  A sample of DNA is digested with a particular restriction enzyme.  The restriction fragments that are produced are placed in one end of an agarose gel.  Electric current is applied to gel.  DNA has a negative charge and is therefore attracted to the positive end of the gel and moves toward it.

26.  Smaller fragments move faster and farther through the gel.  Fragments of the same length travel the same distance and form bands in the gel that are visible when a dye is added.  Identical DNA samples will always yield identical banding patterns when treated with the same restriction enzyme.

27.  Applications:  Useful to compare 2 different DNA samples. Example: can compare 2 alleles for a gene. If they have differences in a restriction site, they will yield different banding patterns on a gel.  Can also recover the DNA from a gel for use.

29.  Can use a technique called Southern Blotting with gel electrophoresis to find bands on the gel containing genes of interest.  Because SO MANY bands are produced when gel electrophoresis is performed on eukaryotic DNA, you need a technique to be able to identify differences – particularly those you are interested in if you are comparing two alleles.

30.  The basics:  Perform gel as usual.  “Blot” gel onto special paper that DNA from gel sticks to.  “Rinse” paper with solution containing probe complementary to gene of interest.  When exposed to film, DNA bound with probe shows up.  Differences in probe binding corresponds to difference in alleles – you can see these differences as differences in banding pattern.  http://highered.mcgraw- hill.com/olc/dl/120078/bio_g.swf http://highered.mcgraw- hill.com/olc/dl/120078/bio_g.swf http://highered.mcgraw- hill.com/olc/dl/120078/bio_g.swf

31.  Noncoding stretches of DNA have differences in nucleotide sequences between individuals just like coding segments of DNA.  These differences are called restriction fragment length polymorphisms or RFLPs.

32.  This is basically the same as a difference in coding sequence – it can also serve as a genetic marker for a particular location in the genome and occurs in many variations in the population and is inherited in a Mendelian fashion.  These can be separated and analyzed using gel electrophoresis and Southern blotting.  This helps in mapping the human genome and in making linkage maps. http://highered.mcgraw- hill.com/olc/dl/120078/bio20.swf http://highered.mcgraw- hill.com/olc/dl/120078/bio20.swf

33.  Mapping a Genome (figuring out the sequence)  Constructing linkage maps using recombination frequencies was first step.

34.  Sequencing DNA using the Sanger Method (page 397)  The following animation shows the Sanger method but with a little more automation as it is done now. It’s the same basic idea.

35. This method uses DNA replication and special nucleotides. These nucleotides stop replication whenever they are added.  This results in many fragments of varying lengths. When these fragments are separated, you can read the sequence from gel.  http://www.pbs.org/wgbh/nova/genom e/sequencer.html http://www.pbs.org/wgbh/nova/genom e/sequencer.html http://www.pbs.org/wgbh/nova/genom e/sequencer.html

36.  Studying Gene Expression (You must read pages 399 & on!)  DNA sequencing can identify regions that are protein coding genes (presence of promoters, etc) but how do we figure out what proteins these code for?  In vitro mutagenesis: mutations are introduced into a cloned gene – reinsert it into a cell and see how phenotype is altered – may help determine the type of protein coded for.

37.  Use DNA microarray assays to determine which genes are being expressed in a particular cell.  http://highered.mcgraw- hill.com/olc/dl/120078/micro50.swf http://highered.mcgraw- hill.com/olc/dl/120078/micro50.swf http://highered.mcgraw- hill.com/olc/dl/120078/micro50.swf

38.  (You need to watch the animation and then read this). cDNA is DNA that is made from mRNA.  Genes that are being expressed are the only genes in a cell making mRNA.  Scientists can isolate mRNA from cells and use reverse transcriptase to produce DNA that must have coded for the mRNA – this is what is called cDNA and what is referred to in the microarray animation.

39.  Applications of DNA technology  Determine evolutionary relationships by comparing genome sequences.  Can also study the variation within our species. We are 99.9% the same as all other humans (our evolutionary history is pretty short). We can look at differences in genes and relate them to human evolution/migratory patterns.

40.  Diagnosis of Disease  Can identify abnormal alleles for early identification of genetic diseases.  Look for HIV RNA in blood and tissue samples

41.  Gene Therapy  Altering a person’s genes (give someone with an abnormal gene the normal gene)  In people with disorders traceable to a single defective gene, it should theoretically be possible to replace or supplement the defective gene with a normal allele. The new allele could be inserted into the somatic cells of the tissue affected by the disorder.  Very little scientifically strong evidence of effectiveness. Even when genes are successfully and safely transferred, their activity typically diminishes after a short period.

43.  Technical questions: How can the activity of the transferred gene be controlled so that cells make appropriate amounts of the gene product at the right time and in the right place?  How can we be sure that the insertion of the gene doesn’t harm some other cell function?

44.  Ethical/Social Questions  Will it inevitably lead to eugenics (a deliberate effort to control the genetic makeup of the human population)?  Should we try to treat human germ-line cells in the hope of correcting a defect in future generations? Should we interfere with evolution in this way? The elimination of unwanted alleles from the gene pool could backfire – genes that are damaging under some conditions may be advantageous under other conditions.

45.  Pharmaceutical Products – using cloning techniques to make insulin, growth hormone, etc. We insert the genes that make these proteins into other organisms so that we can harvest the protein and give it to people.

46.  Environmental Uses  Engineer microbes that can break down heavy metals, toxins in mining sites, oil spills.

47.  Agriculture  Insert human genes into farm animals that produce the protein to harvest – some secrete the desired protein in their milk.  Can make organisms that produce better wool, meat, etc.  Plants with resistance to pesticides and improved nutritional value (rice containing beta carotene needed to make vitamin A)

50. MIT students working with E. coli bacteria inserted a gene into the bacteria that made them smell like mint – just to make their working environment more pleasant.

51. Forensic Evidence  Comparison of samples of body fluids/tissues at crime scene with suspects.  Use probes for RFLPs and gel electrophoresis – provides an individual’s DNA fingerprint. Likelihood that banding pattern on gel will be the same for different people is VERY small because RFLPs are inherited & variable.