2. DNA, RNA and Gel Electrophoresis

3. Central Dogma of Biology
DNA (genetic information in genes) RNA (copies of genes) proteins (functional molecules)
Transcription
Nucleus
Translation
Cytoplasm

4. Structure of DNA
This slide shows the chemical structure of DNA. It is fairly complicated but elegant and I am going to work you through it
DNA is built from a series of nucleotides which are strung together.
One nucleotide consists of a phosphate, sugar, and nitrogenous base
Point out in diagram as purple circle, yellow diamond, and colored squares
The nucleotides are paired left to right and then strung top to bottom to form what looks like a ladder
The phosphate and sugar groups are purely structural and make up the sides of the ladder and are called the sugar-phosphate backbone.
Let’s look at the phosphate group in the middle. It has a central P and four Os and is negatively charged.
Let’s look at the sugar. The sugar is made of five carbons which are numbered 1 – 5 as you can see in this diagram.
The sugar is called deoxyribose because it doesn’t have an oxygen at carbon 2
Let’s look at how the sugar and phosphate connect in this bottom left diagram: You can see that one of the oxygens in the Phosphate group binds to the 5 carbon below it and the other binds to the 3 carbon above it. This connection is called a phosphodiester bond.
Let’s look at how the phosphodiester bonds are formed in the two sides of the ladder. On one side we start with an unattached phosphate group, which then attaches to the carbon 3 etc. while the other one goes in the opposite direction so we say one sside of the ladder goes 3 to 5 prime while the other is antiparrallel and goes 5 to 3 prime.
The nitrogenous bases are in the middle and they carrry the information in genes which we will talk about tomorrow. There are four bases cytosine, guanine, adenine and thymine, abbreviated as CGTA. The connect in what are called base pairs tvia hydrogen bonds o form the rungs of the ladder with C always to G and A always to T
DNAs most stable form is called B and it twists clockwise so is called a double helix.

5. DNA Notes DNA (DNA = deoxyribonucleic acid)
DNA is the genetic material of all living cells and of many viruses.
DNA is: an alpha double helix of two polynucleotide strands.
The genetic code is the sequence of bases on one of the strands.
A gene is a specific sequence of bases which has the information for a particular protein.
DNA is self-replicating - it can make an identical copy of itself.
Replication allows the genetic information to pass faithfully to the next generation.
The chromosomes contain 90% of the cell’s DNA and 10% is present in mitochondria and chloroplasts.
A single unit in the chain is a nucleotide. This consists of a phosphate group, a pentose sugar (D = DNA; R = RNA) and an organic base (ATGC = DNA; AUGC = RNA)
Adenine (A) and Guanine (G) are purine bases and are long.
Thymine (T) and Cytosine (C) are pyrimidine bases are short.
Need one long and one short nucleotide per pair.
Hydrogen bonds link the complementary base pairs:
Two between A and T (A = T) - Three between G and C (G ≡ C)

6. RNA Notes RNA (RNA = ribonucleic acid).
Three different types of RNA, (messenger (mRNA), transfer (tRNA) and ribosomal (rRNA).
All are made in the nucleus (transcription).
ribosomes are synthesized in the nucleolus;
mRNA prepared there too – introns removed
All types of RNA are involved in protein synthesis:
mRNA: copies the information from the DNA.
tRNA: carries the specific amino acid to the mRNA in contact with the ribosome.
rRNA: makes up 55% of ribosomes (the other 45% = protein).

7. Differences between DNA and RNA
DNA is double stranded; RNA is a single stranded.
N.B. ATP is also a nucleotide, with ribose as the pentose sugar.
DNA contains the pentose sugar deoxyribose; RNA contains the pentose sugar ribose.
DNA has the base Thymine (T) but not Uracil (U); RNA has U but not T.
DNA is very long (billions of bases); RNA is short (hundreds to thousands of bases)
DNA is self-replicating, RNA is copied from the DNA so it is not self-replicating
The genetic information is held within the base sequence along a DNA strand. 
A codon is a sequence of three nucleotides, coding for one amino-acid.
The genetic code is universal, thus all life must have had a common ancestor (i.e. evolution)

8. How Does DNA Specify the Sequence of a Protein?
A DNA sequence must be “decoded” to make a protein
This decoding requires creation of an RNA template
Creation of “messenger RNA” is called transcription
Creation of protein from the mRNA is called translation

9. Transcription occurs in the nucleus; Translation occurs in the cytoplasm.

10. Relationship Between Genes and Proteins

12. DNA Replication in Cells (in vivo) النسخ المتماثل للحمض النووي DNA داخل الخلية الحية
DNA replication is the copying of DNA.
It typically takes a cell just a few hours to copy all of its DNA.
DNA replication is semi-conservative (i.e. one strand of the DNA is used as the template for the growth of a new DNA strand).
This process occurs with very few errors (on average there is one error per 1 billion nucleotides copied).
More than a dozen enzymes and proteins participate in DNA replication.

13. DNA Replication This takes place during the S stage of interphase
Nucleotides are synthesized in huge quantities in the cytoplasm.
An enzyme unzips the two complementary strands of DNA.
New complementary nucleotides link to the exposed bases on the separated strands.
The general name for this group of enzymes is DNA polymerase.
A new complementary strand is built along each ‘old’ strand.
Two DNAs, identical to the original and each other, are now present.
Each new DNA molecule is thus ‘half old’ and ‘half new’ ‘semi-conservative replication’.

14. DNA Key enzymes involved in DNA Replication
DNA Polymerase
DNA Ligase
Primase
Helicase
Topoisomerase
Single strand binding protein (RNA)

15. DNA Replication enzymes: DNA Polymerase
catalyzes the elongation of DNA by adding nucleoside triphosphates to the 3’ end of the growing strand
A nucleotide triphosphate is a 1 sugar + 1 base + 3 phosphates
When a nucleoside triphosphate joins the DNA strand, two phosphates are removed.
DNA polymerase can only add nucleotides to 3’ end of growing strand

16. DNA Replication enzymes: DNA Ligase
The two strands of DNA in a double helix are ant parallel (i.e. they are oriented in opposite directions with one strand oriented from 5’ to 3’ and the other strand oriented from 3’ to 5’
5’ and 3’ refer to the numbers assigned to the carbons in the 5 carbon sugar
Given the ant parallel nature of DNA and the fact that DNA polymerases can only add nucleotides to the 3’ end, one strand (referred to as the leading strand) of DNA is synthesized continuously and the other strand (referred to as the lagging strand) in synthesized in fragments (called Okazaki fragments) that are joined together by DNA ligase.

17. Complementary Base-Pairing in DNA
DNA is a double helix, made up of nucleotides, with a sugar-phosphate backbone on the outside of the helix.
Note: a nucleotide is a sugar + phosphate + nitrogenous base
The two strands of DNA are held together by pairs of nitrogenous bases that are attached to each other via hydrogen bonds.
The nitrogenous base adenine will only pair with thymine
The nitrogenous base guanine will only pair with cytosine
During replication, once the DNA strands are separated, DNA polymerase uses each strand as a template to synthesize new strands of DNA with the precise, complementary order of nucleotides.

18. Polymerase Chain Reaction (PCR)
PCR is a much quicker tool for duplicating DNA without cells, developed in 1980s.
PCR is a means to amplify a particular piece of DNA.
Amplify= making many of copies of a segment of DNA.
PCR can make billions of copies of a target sequence of DNA in a few hours.
PCR is a laboratory version of DNA Replication in cells.
The laboratory version is commonly called “in vitro” since it occurs in a test tube while “in vivo” signifies occurring in a living cell.

19. Materials Needed for Extraction
DNA Extraction
1- DNA must be purified from cellular material in a manner that prevents degradation.
2- DNA extraction from plant tissue can vary depending on the material used.
3- Extraction mean breaking down the cell wall and membranes to allow access to nuclear material, with out its degradation.
Materials Needed for Extraction
7- 70% Ethanol.
8- 7.5M Ammonium Acetate.
9- 65o c water bath.
10- Chloroform.
11- Sterile water.
12- Agarose gel.
13- Loading Buffer.
1- CTAB buffer.
2- Microfuge tubs.
3- Mortar and pestle.
4- Liquid Nitrogen.
5- Centrifuge.
6- Absolute Ethanol (Ice cold).

20. Steps for Extraction
1- Liquid nitrogen is employed to break down cell wall material and allow access to DNA.
2- Once the tissue has been sufficiently ground, it can be re-suspended in a suitable buffer, such as CTAB.
3- To purify DNA, insoluble particulates are removed through centrifugation.
4- To remove soluble proteins and other materials, we mixing with chloroform and centrifugation.
5- DNA must be then be precipitated from the aqueous phase and washed thoroughly to remove contaminating salts.
6- To purified DNA, then re-suspended and store in TE buffer or sterile distilled water.
7- To check the quality of the extracted DNA, a sample is run on an agrose gel, stained with Ethidium bromide and visualized under UV light.

21. PCR PCR Thermo-cycler PCR requirements:- 1-The DNA. 2- DNA polymerase.
3- Buffer.
4. Nucleoside tri-phosphates.
5- Primers.
- All of this are placed in a thin-walled tube and then these tubes are placed in the PCR thermal cycler.
PCR Thermo-cycler

22. RNA Reagents Reagents Needed:- DNA sample which you want to amplify.
DNA polymerase.
Taq DNA polymerase – Works at high temps.
Nucleotides , Called (dNTPs).
Pair of primers: One primer binds to the 5’ end of one of the DNA strands, the other primer binds to the 3’ end of the anti-parallel DNA strand.
Water.
Buffer.
All of this are placed in a thin-walled tube and then these tubes are placed in the PCR thermal cycler.

23. The three main steps of PCR
The basis of PCR is temperature changes and the effect that these temperature changes have on the DNA.
In a PCR reaction, the following series of steps is repeated times
(note: 25 cycles usually takes about 2 hours and amplifies the DNA fragment of interest 100,000 fold)
Step 1: Denature DNA:
At 95C, the DNA is denatured (i.e. the two strands are separated)
Step 2: Primers Annealing:
At 40C- 65C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA
Step 3: DNA polymerase Extends the DNA chain:
At 72C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends of the primers.

24. Heat-stable DNA Polymerase
Given that PCR involves very high temperatures, it is imperative that a heat-stable DNA polymerase be used in the reaction.
Most DNA polymerases would denature (and thus not function properly) at the high temperatures of PCR.
Taq DNA polymerase was purified from the hot springs bacterium Thermus aquaticus in 1976
Taq has maximal enzymatic activity at 75 C to 80 C, and substantially reduced activities at lower temperatures.

25. 1:- Denaturation of DNA
This occurs at 95 ºC mimicking the function of helicase in the cell.

26. 2:- Annealing or Primers Binding
Reverse Primer
Forward Primer
Primers bind to the complimentary sequence on the target DNA. Primers are chosen such that one is complimentary to the one strand at one end of the target sequence and that the other is complimentary to the other strand at the other end of the target sequence.

27. 3:- Extension or Primer Extension
DNA polymerase catalyzes the extension of the strand in the 5-3 direction, starting at the primers, attaching the appropriate nucleotide (A-T, C-G)

28. The next cycle will begin by denaturing the new DNA strands formed in the previous cycle.

29. The Size of the DNA Fragment Produced in PCR is Dependent on the Primers
The PCR reaction will amplify the DNA section between the two primers.
If the DNA sequence is known, primers can be developed to amplify any piece of an organism’s DNA.
Forward primer
Reverse primer
Size of fragment that is amplified

30. How PCR Works

31. Gel Electrophoresis of DNA
Gel electrophoresis detects the presence of DNA in a sample
Gel electrophoresis detects the number of nucleotides in a fragment of DNA
e.g., the number of nucleotides in a DNA region which was amplified by PCR
Is a rough estimate, is not exact, need more sophisticated sequencing techniques to get an exact number of nucleotides
Can be used to tentatively identify a gene because we know the number of nucleotides in many genes

32. Equipment Needed Box to hold the gel
Comb to create small wells in the agarose gel to put the DNA sample into at the beginning of the gel
Positive and negative electrodes to create the electrical current
Power supply
Gel photo imaging system
Buffer.

33. Materials
Gel box/tank, gel tray, gel comb(s), power supply, UV viewing table, camera (or more sophisticated gel.
viewing and image producing equipment), pipettes, pipette tips, Kim Wipes, gloves, goggles, 250 ml.
Erlenmeyer flask, graduated cylinder.
Ethidium Bromide (10mg/ml).
Buffer.

34. How Gel Electrophoresis of DNA Works
A sample which contains fragments of DNA is forced by an electrical current through a firm gel which is really a sieve with small holes of a fixed size
Phosphate group in DNA is negatively charged so it is moved towards a positive electrode by the current
Longer fragments have more nucleotides
So have a larger molecular weight
So are bigger in size
So aren’t able to pass through the small holes in the gel and get hung up at the beginning of the gel
Shorter fragments are able to pass through and move farther along the gel
Fragments of intermediate length travel to about the middle of the gel
DNA fragments are then visualized in the gel with a special dye
The number of nucleotides are then estimated by comparing it to a known sample of DNA fragments which is run through the gel at the same time

35. How Gel Electrophoresis of DNA Works

36. The results from a DNA gel
Many samples can be run on one gel- but it is
important to keep track.
• Most gels have one lane as a ‘DNA ladder’ - DNA fragments of known size are used for comparison.