Polynucleotides & DNA

Nucleotides --> Polynucleotides

• The Nucleotides Join Together between the Phosphate group of one nucleotide, and between a Sugar of another. This forms a Phosphodiester Bond (made up of the phosphate group and two ester bonds - Phospho-Di-Ester)
• The chain of Sugars and Phosphates is known as the Sugar-Phosphate Backbone
• Polynucleotides can be Broken Down into Nucleotides again by Breaking the Phosphodiester Bonds.

Double Helix

• Two Polynucleotides join together by Hydrogen Bonding Between the Bases.
• Each base can only bond with a particular partner: this is called Complimentary Base Pairing.
• Adenine (A) always pairs with Thymine (T) and Cytosine (C) always pairs with Guanine (G) (I remember it as the straight letters go together, and the curly letters go together)
• A Pyrimidine always pairs with a Purine.
• 2 Hydrogen bonds form between A-T and 3 Hydrogen Bonds form between G-C
• Two Antiparallel (going in opposite directions) polynucleotide Strands Twist to form the DNA Double Helix

Purifying DNA - Precipitation Reaction

1. Break Up Cells using a blender
2. Make a mixture of washing up liquid, salt, and distilled water, this mixture is called the Detergent
3. Add broken cells to a beaker containing the detergent
4. Put the beaker in a Water Bath (60°C) for 15 Minutes.
5. Now, put the beaker in an Ice Bath, then Filter the mixture.
6. Transfer a small amount of the filtered mixture to a clean boiling tube.
7. Add Protease Enzymes
8. Slowly Dribble some cold ethanol down the side of the boiling tube so it forms a Layer on top of the mixture.
9. Leave it for a few minutes and a White Precipitate should form, which can be removed with a Glass Rod.

Self-Replication

DNA can Copy Itself before cell division

1. DNA  Helicase (Enzyme) Breaks the Hydrogen Bonds Between Two polynucleotide DNA Strands - The helix Unzips.
2. Each Original Strand becomes a Template for a new strand. Free-Floating DNA Nucleotides Join to the exposed bases of each original strand by Complementary Base Pairing.
3. The Nucleotides of the New Strand are joined together by DNA Polymerase (Another Enzyme). This forms the Sugar-Phosphate Backbone. Hydrogen Bonds Form between the bases on the Original and New Strand. It then Twists to form a Double Helix.
4. Each new DNA molecule contains One strand from the Original DNA Molecule and One New Strand

Nucleotides

• A Nucleotide is yet another type of Biological Molecule, it's made from a Pentose Sugar, a Nitrogenous Base and a Phosphate Group.
• All Nucleotides contain the elements, Phosphorus, Oxygen, Nitrogen, Carbon and Hydrogen 

• Nucleotides are the Monomers that make up DNA and RNA
• ADP and ATP are Special types of Nucleotides used to Store and Transport Energy in cells

Deoxyribose (Shown Above)

• The pentose sugar in a DNA nucleotide is 'Deoxyribose'
• Each DNA Nucleotide has the Same Sugar and Phosphate Group, it is the Base on each DNA nucleotide that Varies.
• There are 4 possible bases, Adenine (A), Thymine (T), Guanine (G) and Cytosine (C)
• Adenine and Guanine are a type of base called Purine.
• Thymine and Cytosine are a type of base called Pyrimidine.

• Purine: contains 2 Carbon-Nitrogen Rings joined together
• Pyrimidine: Only 1 Carbon-Nitrogen Ring - so it is Smaller than purine.
• A Molecule of DNA contains Two Polynucleotide Chains - each chain having lots of nucleotides joined together.

Ribose 

• RNA contains Nucleotides with a Ribose sugar instead of deoxyribose.
• An RNA molecule also has a Phosphate group, and one of four different bases.
• However, in RNA, Uracil (U), which is a Pyrimidine, Replaces Thymine as a base.
• An RNA molecule is made up of a Single Polynucleotide Chain

ADP and ATP

• To Phosphorylate a nucleotide, you Add One or More Phosphate Groups to it.
• ADP (Adenosine Diphosphate) contains the base Adenine, the sugar Ribose and Two Phosphate Groups.
• ATP (Adenosine Triphosphate) contains the base Adenine, the sugar Ribose, and Three Phosphate Groups.

ADP, ATP and Energy

• ATP provides Energy for Chemical Reactions in the Cell.
• ATP is Synthesised from ADP and Inorganic Phosphate (Pi) using the Energy From an Energy-Releasing Reaction, (e.g. the breakdown of glucose in respiration.
• The ADP is Phosphorylated to form ATP and a Phosphate Bond is formed.
• Energy is Stored in the Phosphate Bond. When this Energy is Needed by a cell, ATP is Broken Back Down into ADP and Inorganic Phosphate (Pi). The Energy is Released from the Phosphate Bond and Used by the Cell.

Biochemical Tests and Separating Molecules

Biosensors
A Biosensor is a device that uses a Biological Molecule, such as an Enzyme to detect a Chemical.
The biological molecule produces a Signal (i.e. a chemical  signal) which is Converted into an Electrical Signal by a Transducer.
The electrical signal is then processed and can be used to work out other information.

Example:
A Glucose Biosensor is used to determine the Concentration of Glucose in a Solution.
It does this using the enzyme 'Glucose Oxidase' and Electrodes.
The enzyme Catalyses the Oxidation of glucose at the Electrodes. this creates a Charge, which is Converted into an Electrical Signal by the Electrodes (Transducer)
The Electrical Signal is then Processed to work out the Initial Glucose Concentration

Chromatography
The main use is Separating.
Once a solution is separated, we can Identify the Components.
It can be used to Identify Biological Molecules such as Amino Acids, Carbohydrates, Vitamins and Nucleic Acids.
There are loads of different types of chromatography, but we only need to know two, Paper Chromatography and Thin-Layer Chromatography.

Both methods of chromatography have two basic phases:
A MOBILE PHASE

• Where the Molecules Can Move
• in both paper and thin-layer chromatography, the Mobile Phase is a Liquid Solvent, such as Ethanol or Water.

A STATIONARY PHASE

• Where the Molecules Can't Move
• In Paper chromatography, the stationary phase is a piece of Paper.
• In Thin-Layer Chromatography, the stationary phase is a Thin (<0.5mm) Layer of a Solid, i.e. Silica Gel, Glass or Plastic.

They both use the same basic method:

1. The Mobile Phase moves Through or Over the Stationary Phase, 
2. The Components in the mixture spend Different amounts of Time in the Mobile phase And the Stationary phase.
3. The Components that spend Longer in the Mobile phase travel Faster or Further.
4. The time spent in the different phases is what separates out the components of the mixture.

PAPER CHROMATOGRAPHY

1. Draw a pencil line near the bottom of the chromatography paper and put a concentrated spot of the mixture of amino acids on it.
2. Add a small amount of prepared Solvent in a beaker and Dip the bottom of the paper into it. Cover it with a lid to stop the solvent evaporating.
3. As the solvent spreads up the paper, the different amino acids move with it, but at different rates, so they separate out.
4. When the solvent's nearly reached the top, Take the paper Out and Mark the Solvent Front (the highest point the solvent has reached), now leave the paper to Dry before analysing it.
5. Since amino acids are colourless, Spray them with Ninhydrin solution to turn the amino acids purple, then use the Rf values to Identify the separated molecules.

Colorimetry

Colorimetry is used to determine the Concentration of a Glucose Solution.

• To do this, we need to use Benedict's Reagent and a Colorimeter to get an Estimate of how much Reducing Sugar there is in a solution.
• A Colorimeter measures the strength of a coloured solution by detecting how much Light Passes Through it, the More Concentrated the colour, the Higher the Abundance of Reducing Sugars

PAST PAPER QUESTION!

Describe how the concentration of a reducing sugar can be measured using a colorimeter?

Using Known Concentrations of reducing sugar (1)

Heat with Benedict's Solution at least 80°C (1)

The Colour Changes to  green, yellow, orange, brown or (brick) red (1)

Then press zero on the colorimeter by using  a Blank Cuvette With Benedict's solution. (1)

Then take a Reading of Transmission (how much light passes through the cuvette) of the Solution. (1)

Plot a Calibration Curve for Transmission against Reducing Sugar Concentration then use the reading of the Unknown Sugar Solution and read off graph to find the Concentration of the Unknown sugar solution (1)

Biochemical Tests for Molecules

This is just loads of practicals involving biological molecules we need to know how to do.

Benedict's Test for Sugars
Sugar is a general term for Monosaccharides and Disaccharides. All sugars can be classed as Reducing or Non-Reducing. The Benedict's Test Differs depending on the Type of Sugar you are testing for.

BENEDICT'S TEST FOR REDUCING SUGARS

• Reducing sugars include All Monosaccharides (i.e. glucose) and some Disaccharides (i.e. maltose and lactose)
• To the sample, we need to add Benedict's Reagent (It's blue).
• now we need to heat it up and Bring it to the Boil. This is usually done in a Water Bath.
• The colour should go: BLUE>GREEN>YELLOW>ORANGE>BRICK-RED
• If the test is Positive, the Precipitate will Change Colour. The Higher the Concentration of reducing sugars, the Further the Colour Change will go.
• We can use this to compare the amount of reducing sugar in different solutions, however, weighing would be a more reliable alternative.

BENEDICT'S TEST FOR NON-REDUCING SUGARS

• If the result of the above test is negative, it could still have non-reducing sugars in the solution, such as sucrose. But first, we have to Break Them Down into Monosaccharides.
• For this, you will need a New Sample of the Test Solution.
• Add Dilute Hydrochloric Acid and Heat It in a water bath that has been Brought to the Boil.
• Then Add Sodium Hydrogencarbonate to neutralise the solution. Then carry out Benedict's test for Reducing Sugars as explained above. 
• If the solution forms a precipitate which is not blue, the solution contains non-reducing sugars.
• If the solution remains blue, there are no sugars present.

Test Strips for Glucose

Glucose can be tested for using Test Strips coated in a Reagent. The strips are Dipped in a Test Solution and Change Colour if Glucose is Present. The test strip can be Compared to a Chart to Estimate the Concentration of Glucose present. This is used in Urine Tests, which may indicate Diabetes.

Iodine Test for Starch

• Add iodine dissolved in Potassium Iodide solution to the test sample.
• If Starch is Present, the sample will change from brown/orange to Blue/Black.
• If there's no starch, the sample will stay brown/orange

Biuret Test for Proteins.

There are 2 stages:

1. The test solution must be alkaline, so we need to add a few drops of Sodium Hydroxide solution.
2. Then add Copper (II) Sulphate solution.

If Proteins are Present, the solution turns purple.

If no proteins are present, the solution will stay blue.

Emulsion Test for Lipids

• Add Ethanol to the Test Sample, and Shake Well for 60s.
• Then pour the solution into a test tube of Water.

If Lipids are Present, the solution will turn Milky.

If no lipids are present, the solution will remain clear.

Inorganic Ions

• An Ion is an Atom (or group of atoms) with an Electric Charge
• An Ion with a Positive charge is a Cation
• An Ion with a Negative charge is an Anion
• An Inorganic Ion is one that Doesn't Contain Carbon (mostly!)

We need to know about 5 cations and 5 anions:

Name of Ion	Chemical Symbol	Examples of roles in biological processes.
Calcium	Ca2+	The Transmission of Nerve Impulses and the Release of Insulin from the pancreas. It acts as a Cofactor for many enzymes. Also important in Bone formation
Sodium	Na+	For Generating Nerve Impulses for Muscle Contraction and for Regulating Fluid Balance in the body.
Potassium	K+	Generating Nerve Impulses, for Muscle Contraction and Regulating Fluid Balance. It activates Enzymes needed for Photosynthesis in plants
Hydrogen	H+	Affects the pH of Substances  if the amount of H+ is greater than the amount of OH- then it is an acid, vice versa is an alkali. Also Important for Photosynthesis Reactions in the thylakoid membranes inside chloroplasts
Ammonium	NH4+	Absorbed from the Soil by the plants and is an important Source of Nitrogen (used to make amino acids and nucleic acids)
Nitrate	NO3-	Exactly the same as NH4+
Hydrogencarbonate	HCO3-	Acts as a Buffer to help maintain the pH of the Blood
Chloride	Cl-	Involved in the 'Chloride Shift' which helps to Maintain pH of the Blood during Gas Exchange. Acts as a Cofactor for Amylase (an enzyme). Also involved in some Nerve Impulses
Phosphate	PO43-	Involved in Photosynthesis and Respiration Reactions. It is needed for the Synthesis of many Biological Molecules such as Nucleotides including ATP, Phospholipids and Calcium Phosphate (strengthens bones)
Hydroxide	OH-	Affects the pH of Substances (see H+)