2.  Molecular biology; the study of biology at the molecular level.  Molecular biology; the study of gene structure and functions at the molecular level to understand the molecular basis of hereditary, genetic variation, and the expression patterns of genes.

3.  The structure of DNA was described by British Scientists Watson and Crick as long double helix shaped with its sugar phosphate backbone on the outside and its bases on inside; the two strand of helix run in opposite direction and are anti-parallel to each other. The DNA double helix is stabilized by hydrogen bonds between the bases.  This structure explains how genes engage in replication, carrying information and acquiring mutation.  The G+C content of a natural DNA can vary from 22-73% and this can have a strong effect on the physical properties of DNA, particularly its melting temperature.

4.  DNA and RNA are long chain polymers of small compound called nucleotides.  Each nucleotide is composed of  a base;  sugar (ribose in RNA or deoxyribose in DNA) and  a phosphate group. General Structure of Nucleic Acid

5.  There are four different types of nucleotides found in DNA, differing only in the nitrogenous base : A is for adenine; G is for guanine; C is for cytosine and T is for thymine.  These bases are classified based on their chemical structures into two groups: adenine and guanine are double ringed structure termed purine, thymine and cytosine are single ring structures termed pyrimidine.

6.  The bases pair in a specific way:  Adenine A with Thymine T (two hydrogen bonds) and  Guanine G with Cytosine C (three hydrogen bonds).

7. Strands in the DNA runs antiparallel

8. FORM OF DNA CharacteristicA-DNAB-DNAZ-DNA diameter (D)2.6 nm2.0 nm1.8 nm bp/turn1110.412 degrees rotation/bp+32.7+34.6-30.0 axial distance/turn2.8 nm3.4 nm4.5 nm axial distance between bp0.25 nm0.33 nm0.38 nm

10. Replication proceeds in a semiconservative manner, each strand of the DNA helix serves as a template for the synthesis of complementary DNA strands. This lead to the formation of two complete copies of the DNA molecule, each consisting of one strand derived from the parent DNA molecule and one newly synthesized complementary strand.

11. Eukaryotic genes: DNA molecules complexed with other proteins especially basic proteins called histones, to form a substance known as chromatin.

12.  Eukaryotic chromatin is folded in several ways. The first order of folding involves structures called nucleosomes, which have a core of histones, around which the DNA winds ( four pairs of histones H2A, H2B,H3 and H4 in a wedge shaped disc, around it wrapped a stretch of 147 bp of DNA).

13.  Deoxyribonucleic Acid (DNA), the gigantic molecule which is used to encode genetic information for all life on Earth excepts some viruses.  The chemical basis of hereditary and genetic variation are related to DNA.  DNA directs the synthesis of RNA which in turn directs protein synthesis.

14. Central Dogma of Molecular Biology The flow of genetic information as follows: The flow of genetic information as follows:

15.  To understand the basic process of isolation of DNA from various sources eg blood, tissue, bacteria.  To realise that different types of DNA require different methods of isolation.  To realise that the method used is dependent upon the final application.  To understand the basis of gel electrophoresis  To realise that there are different types of gel electrophoresis. MG331/MB331

17. Introduction Deoxyribonucleic acid (DNA) isolation is an extraction process of DNA from various sources. Methods used to isolate DNA are dependent on the source, age, and size of the sample. Despite the wide variety of methods used, there are some similarities among them. In general, they aim to separate DNA present in the nucleus of the cell from other cellular components. Isolation of DNA is needed for genetic analysis, which is used for scientific, medical, or forensic purposes. Scientists use DNA in a number of applications, such as introduction of DNA into cells and animals or plants, or for diagnostic purposes. In medicine the latter application is the most common. On the other hand, forensic science needs to recover DNA for identification of individuals (for example rapists, petty thieves, accident, or war victims), paternity determination, and plant or animal identification

18. Sources for DNA isolation are very diverse. Basically it can be isolated from any living or dead organism. Common sources for DNA isolation include whole blood, hair, sperm, bones, nails, tissues, blood stains, saliva, buccal (cheek) swabs, epithelial cells, urine, paper cards used for sample collection, bacteria, animal tissues, or plants.

19. How Can We Recover DNA From a Variety of Sources of Biological Evidence? Blood Semen Saliva Urine Hair (w/Root & Shaft) Teeth Bone Tissue Cigarette Butts Envelope & Stamps Fingernail Clippings Chewing Gum Bite Marks Feces

20. 1. Maximize DNA recovery 2. Remove inhibitors 3. Remove or inhibit nucleases 4. Maximize the quality of DNA

21.  Organic (Phenol-Chloroform) Extraction  Non-Organic (Proteinase K and Salting out)  Chelex (Ion Exchange Resin) Extraction  FTA  Paper (Collection, Storage, and Isolation) The method utilized may be sample dependant, technique dependant, or analyst preference

22.  Genomic DNA  SDS/Proteinase K  Qiagen columns  Alkaline method  Automated methods  Plasmid DNA  Alkaline/SDS  Qiagen column methods  Bacteriophage M13 DNA  PEG precipitaton method  Bacteriophage lambda DNA  PEG/Salt precipitation method MG331/MB331

23.  Cell extraction  Organic - phenol, CHCl 3  high salt  guanidinium HCl  Removal of cell debris  proteins, lipids, polysaccharides  Concentration of DNA  ethanol, isopropranol  DNA absorbing matrix  CTAB, spermidine  Optional steps  Rnase A removal of RNA MG331/MB331

24.  Perhaps the most basic of all procedures in forensic molecular biology is the purification of DNA. The key step, the removal of proteins, can often be carried out simply by extracting aqueous solutions of nucleic acids with phenol and/or chloroform.  Presence of proteins, lipids, polysaccharides and some other organic or inorganic compounds in the DNA preparation can interfere with DNA analysis methods, especially with polymerase chain reaction (PCR). They can also reduce the quality of DNA leading to its shorter storage life

25.  Cell Lysis Buffer - lyse cell membrane, nuclei are intact, pellet nuclei.  Resuspend nuclei, add Sodium Dodecly Sulfate (SDS), Proteinase K. Lyse nuclear membrane and digest protein.  DNA released into solution is extracted with phenol-chloroform to remove proteinaceous material.  DNA is precipitated from the aqueous layer by the additional of ice cold 95% ethanol and salt  Precipitated DNA is washed with 70% ethanol, dried under vacuum and resuspended in TE buffer.

26.  Cell Lysis Buffer - Non-ionic detergent, Salt, Buffer, EDTA designed to lyse outer cell membrane of blood and epithelial cells, but will not break down nuclear membrane.  EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as Mg2+. Mg2+is a cofactor for Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are inactivated.

27.  Proteinase K - it is usual to remove most of the protein by digesting with proteolytic enzymes such as Pronase or proteinase K, which are active against a broad spectrum of native proteins, before extracting with organic solvents. Protienase K is approximately 10 fold more active on denatured protein. Proteins can be denatured by SDS or by heat.

28. To obtain DNA in a relatively purified form which can be used for further investigations, i.e. PCR, sequencing, etc

29. Break down the cell wall and membranes Centrifuge to separate the solids from the dissolved DNA Precipitate the DNA using isopropanol Centrifuge to separate the DNA from the dissolved salts and sugars Wash the DNA pellet with Ethanol and dry the pellet Dissolve DNA Overview of DNA Extraction

30.  Most DNA extraction protocols consist of two parts 1. A technique to lyse the cells gently and solubilize the DNA 2. Enzymatic or chemical methods to remove contaminating proteins, RNA, or macromolecules  In plants, the nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify the DNA from the rest of the cell. 1. Lysis 2. Precipitation 3. Wash 4. Resuspension

31.  The cell wall (made of cellulose) is disrupted by mechanical force (for example, grinding the leaves)  Then the addition of a detergent in the which breaks down the cell membranes  Detergents are able to disrupt membranes due to the amphipathic (having both hydrophilic and hydrophobic regions) nature of both cellular membranes and detergent molecules. The detergent molecules are able to pull apart the membranes  The end result of LYSIS is that the contents of the plant cells are distributed in solution.

32.  In a research lab, the first part of precipitation uses phenol/chloroform to remove the proteins from the DNA  Phenol denatures proteins and dissolves denatured proteins.  Chloroform is also a protein denaturant  The second part of research lab DNA precipitation is the addition of salts  The salts interrupt the hydrogen bonds between the water and DNA molecules.  The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol  In the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution.  The DNA is pelleted by spinning with a centrifuge and the supernatant removed.

33. Washing: The precipitated DNA is laden with acetate salts. It is “washed” with a 70% ethanol solution to remove salts and other water soluble impurities but not resuspend the DNA. Resuspension: The clean DNA is now resuspended in a buffer to ensure stability and long term storage. The most commonly used buffer for resuspension is called 1xTE (The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation) EDTA further inactivates nucleases, by binding to metal cations required by these enzymes Washing and Resuspension:

34.  The product of your DNA extraction will be used in subsequent experiments  Poor quality DNA will not perform well in PCR  You will want to assess the quality of your DNA extraction using the following simple protocol:  Mix 10 µL of DNA with 10 µL of loading buffer  Load this mixture into a 1% agarose gel  Analyze results (the following slides provide guidance)

35.  Polyacrylamide gel electrophoresis  20bp - 2000bp  Conventional agarose gel electrophoresis  300bp - 40,000bp  100bp-2000bp (special agaroses)  low melting point agaroses  Pulse field/CHEF  40kbp - 2000kbp MG331/MB331

36. If properly done, genomic extraction should result in bright bands in the very high base pair range of a gel electrophoresis. Sizes of Genomic DNA for various Species in kbp E. Coli 4,640,000bp Yeast 12,100,000bp Fruit Fly 140,000,000bp Human3,000,000,000bp Pea4,800,000,000bp Wheat 17,000,000,000bp The genomic fragments run at ~12kbp because they are sheared during extraction Analyzing DNA Samples in a Research Lab

37. Nucleic Acid Analysis via UV Spectrophotometry By measuring the amount of light absorbed by your sample at specific wavelengths, it is possible to estimate the concentration of DNA and RNA. Nucleic acids have an absorption peak at ~260nm. [dsDNA] ≈ A 260 x (50 µg/mL) [ssDNA] ≈ A 260 x (33 µg/mL) [ssRNA] ≈ A 260 x (40 µg/mL) DNA Absorption Spectra

38. THANK YOU