1. Studying Genomes Learning Outcome Outline the steps involved in sequencing the genome of an organism.

2. Cloning the chromosome section The chromosome is physically sheared into sections. Existing approximate chromosome maps allow identification of the location that each section has come from The sections are incorporated into BACs (Bacterial Artificial Chromosomes) A BAC is a modified bacterial plasmid that incorporates the foreign DNA. The foreign DNA is flanked by STCs (Sequence Tag Connectors) that act as markers to identify the incorporated DNA The BAC is inserted into a bacterium (using electroporation) and the whole BAC is copied each time the bacteria reproduce giving numerous clones of the DNA being investigated. These cells are called clone libraries.

3. Sequencing the BAC section (step one) A number of copies of the BAC section are extracted from the cloned bacteria Next the DNA is treated with restriction enzymes or endonucleases

4. Restriction enzymes Restriction enzymes work by cutting the DNA at specific sequences, often in a staggered way, to leave sequences of unpaired bases called sticky ends. If the cut is not staggered it leaves blunt ends. DNA ligase is used to stick the DNA sugar phosphate back bones back together Hydrogen bonds between the complementary bases hold them together

5. Sequencing the BAC section (step two) Once the fragments of DNA have been produced using restriction enzymes these can be separated by using electrophoresis. When the fragments are put into the agarose gel with an electric current across, the smallest fragments move through the gel fastest.

6. Learning outcome Outline how DNA fragments can be separated by size using electrophoresis.

7. Electrophoresis DNA fragments are placed in wells in the agarose gel at the cathode end Gel is immersed in buffer and an electric current passed through DNA has –ve charge due to many phosphoryl groups so is attracted to positive electrode (anode) Short DNA fragments diffuse faster through the gel. Position of fragments is revealed by using a dye that stains DNA Fragments can be lifted from gel using “Southern blotting” Fragments can be further sequenced using PCR and Chain Termination Or probed to see if they contain mutations

8. Electrophoresis sites to look at http://www.sumanasinc.com/webcontent/a nimations/content/gelelectrophoresis.htmlhttp://www.sumanasinc.com/webcontent/a nimations/content/gelelectrophoresis.html http://www.dnalc.org/resources/animations /gelelectrophoresis.html

9. Sequence or probe? Once the DNA fragments have been separated you can either: See if any of the fragments contains the DNA sequence you are looking for by using a probe Or Determine the sequence of bases using chain termination Both techniques need to be preceded by use of the polymerase chain reaction PCR

10. Learning outcome Outline how the polymerase chain reaction can be used to make multiple copies of DNA fragments.

11. Polymerase Chain Reaction (PCR) Remove double stranded DNA from gel by southern blotting Heat DNA to 95 degrees C to separate strands Add primers (short bits of DNA for DNA polymerase to bind to) Reduce temp to 55 degrees. Primers anneal (bind to complementary bases) Raise temp to 72 degrees and add DNA polymerase DNA polymerase binds and makes new complementary DNA using free nucleotides Repeat the process to increase DNA exponentially

12. PCR websites to look at http://lifesciences.envmed.rochester.edu/ movies/PCR_final.swfhttp://lifesciences.envmed.rochester.edu/ movies/PCR_final.swf http://www.sumanasinc.com/webcontent/a nimations/content/pcr.htmlhttp://www.sumanasinc.com/webcontent/a nimations/content/pcr.html NB Make sure you understand why the DNA polymerase used for this process is called “Taq” polymerase

13. Learning outcome Describe how DNA probes can be used to identify fragments containing specific sequences.

14. The Action of DNA Probes Single stranded DNA from PCR or electrophoresis can be investigated using probes These are short single stranded pieces of DNA complementary to the piece of DNA being sought. The probe is labelled with a radio-active or fluorescent marker When copies of the probe are added to samples they will anneal to any complementary strands Fogging of photographic film or fluorescing under UV light shows areas where the DNA sequence being sought is present

15. Microarrays for Genetic Testing Microarrays can be made with many probes fixed to a “chip” Probes are made that have complementary sequences to those found in faulty alleles for genetic diseases The test DNA fragments are applied to the microarray If fragments anneal to the probes this indicates that the person’s DNA is carrying the faulty alleles and they may develop the genetic disease Analysis is by comparing test DNA with the annealing and fluorescence patterns of known fragments

16. Automated sequencing of DNA using chain termination (outline the steps involved in sequencing the genome of an organism) This method uses dideoxynucleotide triphosphates(ddNTPs) in addition to normal nucleotides. The ddNTPs have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs) Dideoxynucleotides are chain terminators as there is no OH group to contribute to the condensation reaction that is required for the next nucleotide to add to the chain. Terminators are added randomly to the growing chains giving a range of fragment lengths. These are then passed through an electrophoresis gel and sequenced by laser

17. Suggested websites with animations about Automated DNA sequencing http://www.pbs.org/wgbh/nova/genome/sequenc er.html#http://www.pbs.org/wgbh/nova/genome/sequenc er.html# http://www.dnalc.org/resources/animations/cycs eq.htmlhttp://www.dnalc.org/resources/animations/cycs eq.html http://www.wiley.com//college/pratt/0471393878/ student/animations/dna_sequencing/index.html All of these are excellent The last of these websites goes through the whole process step by step explaining each part and has review questions to check your understanding. I recommend it!

18. Stretch and Challenge RNA interference The presence of double stranded RNA in a cell initiates the action of an enzyme called “Dicer” Dicer cuts the dsRNA up and makes small inhibitory RNA (siRNA) siRNA complexes with several proteins to produce RISC (RNAi silencing complex) RISC unwinds the double strand siRNA then binds to the active mRNA that should code for a polypeptide during translation “slicer” enzyme chops the mRNA and degrades its nucleotides

19. Using RNA interference as a contraceptive Prior to fertilisation mature ova make ZP3 protein that allows sperm to bind to and penetrate the membrane No ZP3 protein =no fertilisation Introduce dsRNA with a section complementary to mRNA for ZP3 Dicer chops dsRNA to produce siRNA siRNA complexes to form RISC RISC unwinds dsRNA siRNA binds to mRNA for ZP3 Slicer chops and degrades mRNA so no ZP3 is made and no fertilisation takes place!

20. Outline how gene sequencing allows for genome wide comparisons between individuals and species. See page 167 of your text book Make notes on it for yourself!

21. Questions! Make sure you can answer all the questions on pages 166 to 173! Learn the stages in electrophoresis techniques Make sure you could explain how PCR is used in forensic investigations Why isn’t the DNA polymerase used in PCR denatured at high temperatures?

22. A whole website dedicated to biology animations etc! http://bio-alive.com/animations/DNA.htm