Sequencing

 http://www.dnalc.org/ddnalc/resources/cycseq.html

http://www.dnai.org/text/mediashowcase/index2.html?id=552

http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf

Linkage mapping

http://courses.bio.psu.edu/fall2005/biol110/tutorials/tutorial8.htm

Animations - Evolution

http://www.echalk.co.uk/Science/Biology/PepperedMoth/Peppered_MothWEB.swf

(Peppered Moth Game)

http://www.johnkyrk.com/evolution.html

(Interesting overview of evolution)

http://www.wwnorton.com/college/biology/discoverbio3/core/content/index/animations.asp

(Various Animation Links)

http://trc.ucdavis.edu/biosci10v/bis10v/media/ch13/archipelago.html

(Allopatric Speciation)

http://www.pbs.org/wgbh/evolution/darwin/origin/index.html

(Adapative Radiation)

http://trc.ucdavis.edu/biosci10v/bis10v/media/ch12/finches.html

(Adaptive Radiation)

http://bcs.whfreeman.com/thelifewire/content/chp24/2402001.html

(Speciation)

Animations - Homeostasis

http://pennhealth.com/health_info/animationplayer/homeostasis.html

http://trc.ucdavis.edu/biosci10v/bis10v/week10/07homeostasis.html

Animations – Cell Signalling

http://entochem.tamu.edu/G-Protein/index.html

http://www.wiley.com/college/pratt/0471393878/student/animations/signal_transduction/index.html

http://www.wiley.com/legacy/college/boyer/0470003790/animations/signal_transduction/signal_transduction.htm

http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/moaction/surface.html

http://home.earthlink.net/~shalpine/anim/Life/memb.htm

http://telstar.ote.cmu.edu/Hughes/tutorial/cellmembranes/orient2.swf

http://telstar.ote.cmu.edu/biology/downloads/membranes/index.html

http://www.stolaf.edu/people/giannini/flashanimat/lipids/membrane%20fluidity.swf

http://www.microscopy-uk.org.uk/index.html?http://www.microscopy-uk.org.uk/amateurs/movigifs.html

http://microscope.mbl.edu/baypaul/microscope/animations/rarbur/rarbur_01.htm

http://www.bioscope.org/taste/cd1/a0255.htm

Topic: Proteins
No.	Web-links
1	http://www.bbc.co.uk/education/asguru/biology/02biologicalmolecules/01proteins/12polymers/index.shtml

Topic: Enzymes
No.	Web-links
1	http://www.stolaf.edu/people/giannini/flashanimat/enzymes/prox-orien.swf
2	http://resources.emb.gov.hk/biology/english/virtual_lab/flash/enzyme_lab.html
3	http://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swf
4	http://www.northland.cc.mn.us/biology/Biology1111/animations/enzyme.swf

Common Mistakes & Misconceptions in Biology  

Cell Structure and Cell Membrane

l   Cisterna ≠ crista

Cisterna = lumen of RER or Golgi Apparatus

Crista (cristae= plural) = infolding of the inner mitochondrial membrane

Wrong	Correct
Ions are polar…..	Ions are charged hence they cannot pass through the hydrophobic core of the Cell Surface Membrane.
Nucleolus contains rRNA	Nucleolus synthesizes rRNA (which forms part of ribosome), but it itself is made of DNA (coiled around histones)
Fatty acid/ hydrocarbon tails interact with hydrophobic bonds	Fatty acid/ hydrocarbon tails interact with hydrophobic interactions
Ribosomes have one membrane	Ribosomes do not have membranes
Facilitated diffusion uses only channel proteins. Active transport uses only carrier proteins.	Facilitated diffusion uses both channel protein and carrier proteins. The carrier protein can change conformation without ATP hydrolysis. Active transport uses only carrier proteins which can change conformation only upon ATP hydrolysis. (Campbell 8th edition pg 135). Once, Cambridge question goes against this rule (see 2006/P2/Q1) but you still have to answer the question based on diagram.
Passage of small, hydrophobic molecules through the phospholipid bilayer is called “diffusion”	Passage of small, hydrophobic molecules through the phospholipid bilayer is called “simple diffusion”
Exocytosis is the same as active transport	Exocytosis is a type of bulk transport requiring vesicles, while active transport uses carrier proteins.

Biological Molecules

Wrong	Correct
Cellulose is a protein OR collagen is a polysaccharide	Cellulose is a polysaccharide AND collagen is a (fibrous) protein
Collagen is made up of triple helix	Tropocollagen is made up of triple helix.
Globular protein is globular in shape	Globular protein is spherical in shape. Use synonyms!

Enzymes

l   Active site can only be used when describing enzymes.

For other proteins eg. Receptors, pumps, transcription factors, use allosteric sites / binding sites.

l   Failure to name the types of bonds affected when subjected to denaturation agents/ factors. E.g. Candidates must mention hydrogen/ionic bonds when high temperature is applied, ionic bonds when pH is changed or heavy metals added, hydrophobic interactions when organic solvents added and disulphide bonds when a reducing agent is added.

l   Denaturation has two meanings:

-       For proteins: disruption of tertiary structure, causing the protein to lose its 3D conformation

-       For DNA : breaking of hydrogen bonds between complementary base pairs causing the two DNA strands to separate

Cell and Nuclear Division

l   Chromatids separate to become chromosomes

Chromatids are no longer called chromatids after they separate from the centromere

Wrong	Correct
Chromosomes are pulled to opposite ends of the cells by spindle fibres	Chromosomes are pulled to opposite poles of the cells by spindle fibres
Centromere split	Centromere divide
Chromosome replicate	DNA replicate (so each chromosome is now seen comprising of two sister chromatids)
Mitosis has no homologous chromosomes	Homologous chromosomes are already in cells irregardless of whether they are undergoing cell and nuclear division. All humans have 23 pairs of homologous chromosomes.
Diploid number = amount of DNA	Diploid number is the total number of chromosomes. DNA amount is twice in a chromosome with 2 sister chromatids compared to a chromosome that does not have sister chromatids.
DNA replication occurs in the interphase between meiosis I and II	DNA replication occurs once only in the interphase before meiosis I
Organism is sterile because gametes have odd number of chromosomes	Organism is sterile because the organism itself have odd number of chromosomes and/or have even number of chromosomes that do not occur in homologous pairs

DNA and Genomics

l   Unzip ≠ unwind

Unzip = breaking of hydrogen bonds between complementary base pairs to separate the two DNA strands.

Unwind = untwisting of the DNA double helix

l  

Be careful how you use the 2 terms!

1 DNA strand = 1 polynucleotide chain

l   1 DNA molecule is made up of 2 DNA strands

l   The triplet of 3 bases that forms the genetic code is known as

Base triplet in DNA

Codon in mRNA (some textbooks refer codon to DNA too, but we’ll keep to mRNA for now)

Anticodon in tRNA

Wrong	Correct
Non-coding genes	Non-coding sequences/ regions Genes are already defined as coding DNA
In leading strand, DNA is synthesized in the 5’ to 3’ direction, while in lagging strand, DNA is synthesized in the 3’ to 5’ direction	DNA polymerase III synthesizes DNA daughter strands in 5’ to 3’ direction only. Leading strand is formed due to replication towards replication fork, while the lagging strand is formed due to replication away from replication fork.

Control of Eukaryotic and Prokaryotic Genome

Wrong	Correct
Control elements are proteins/ transcription factors are DNA	Control elements (promoter, silence and enhancer) are non-coding DNA sequences. Transcription factors (which are proteins) bind to control elements to regulate transcription.
Prokaryotes have control elements	Eukaryotes have control elements, while prokaryotes have regulatory sequences (like operator) and regulatory genes (like lacI gene).
Bacteria divides by mitosis	Bacteria divides by binary fission.

Energetics

Wrong	Correct
One of the electron carriers in the electron transport chain is a proton pump.	The electron carriers in the electron transport chain are coupled to proton pumps.
Electrochemical gradient is the same as concentration gradient	Electrochemical gradient includes  concentration gradient AND electric potential gradient (so be careful how you use the term!)
When Cl- ions enter the neurone, it leads to repolarization.	When Cl- ions enter the neurone, it leads to hyperpoloarization.
Light dependent reactions occur in the presence of light while light independent reaction occurs in the dark	Light dependent reactions occur in the presence of light while light independent reaction can occur in the presence and absence of light.

Genetic Basis of Variation

Wrong	Correct
The two genes are linked so closely together that crossing over does not happen.	The two genes are linked so closely together that crossing over to separate the two genes does not happen.

Diversity and Evolution

                            

Wrong	Correct
Traits are passed on to the next generation	Genes/alleles are passed on to the next generation
Organisms mutate to adapt to the environment	Mutations are random, they cause variation within populations
Mutations occur at a constant rate in neutral theory	Neutral mutations have been observed/ found to occur at a constant rate in certain DNA sequences or genes
Only silent mutation is involved in neutral theory	Neutral theory involves silent mutation and any mutation that results in a phenotype which is selectively neutral.

Applications

Wrong	Correct
Gene therapy is a technique for introducing healthy/ therapeutic gene	…..introducing normal allele/gene
Package the normal allele into liposomes/ virus	Package the DNA containing the normal allele/gene into liposomes/ virus
Deletion of a codon results in phenylalanine not to be produced/ gene lacking the amino acid	Deletion of a codon results in phenylalanine not to be coded/ protein lacking the amino acid
Insertional inactivation can be used to distinguish recombinant plasmids from reannealed plasmids	Insertional inactivation can be used to distinguish bacteria containing recombinant plasmids from bacteria containing reannealed plasmids.
In plant cloning, clones have same genes/ genetically similar to the same parent	In plant cloning, clones are genetically identical to the same parent
Stem cells repair damaged cells	Stem cells replace damaged cells Stem cells repair damaged organs
In autoradiography, expose the nitrocellulose membrane to X-ray	In autoradiography, place a piece of X-ray film over the nitrocellulose membrane, which will reveal the bands
Southern Blotting includes gel electrophoresis	Southern Blotting is carried out AFTER gel electrophoresis.

l   When describing gel electrophoresis, write “ direct current was applied”

l   In nucleic aid hybridization: if radioactive probe is used, autoradiography is used to visualise the DNA bands on X-ray film

l   In gel electrophoresis: If ethidium bromide is use, UV light is used to visualise the bands on the gel

l   DNA ligase forms phosphodiester bonds

l   Restriction enzymes breaks/cleave phosphodiester bonds. Always write, restriction enzyme cuts (not cleave, degrade, break down) DNA at restriction sites

l   Ligation = formation of phosphodiester bonds between nucleotides

l   Annealing = formation of hydrogen bonds between complementary base pairs

l   Southern Blotting simply means putting the gel into an alkali so the alkali can denature the DNA into single-strands. Place a nitrocellulose/ nylon membrane over the gel under heavy weight so that the single-stranded DNA can bind to the membrane after travelling by capillary action.

l   Nucleic acid hybridization simply means complementatary base pairing through hydrogen bonds between single-stranded, radioactive and specific probe and single-stranded DNA.

l   Autoradiography simply means placing a piece of photographic or X-ray film over the nitrocellulose membrane, which will reveal the bands.

l   How to use “RFLP” term? This can be used in e.g. ….perform RFLP analysis…/… if RFLP band fragment is seen…/… RFLP locus is located so close to disease allele…

Synapsis
Crossing-over
Chiasmata
Bivalents
THREE Sources of Variation
Crossing Over at Prophase I
Crossing Over at Prophase I
Independent Assortment at Metaphase I
Independent Assortment at Metaphase I
Possible combinations = 2n, n = haploid number.
Humans: n = 23.
Possible combinations = 223 = 8.4 million!
Independent Assortment is also known as Mendel’s Second Law
Random Fertilization
Each Gamete
 One out of about 8.4 million (223) possible chromosome combinations due to independent assortment.
2 gametes fuse
 Zygote with any of about 70 trillion (223 X 223) diploid combinations!!
Mendel’s First Law
Law of Segregation

A somatic cell carries two alleles at any one locus.
Alleles of a gene pair segregate during anaphase I of meiosis
Half of the gametes carry one allele of a gene pair; the other half carry the other allele.
Summary of meiosis and mitosis
Assignment
Explain the need for the production of genetically identical cells & fine control of replication

Since the functions of mitosis are to produce new cells for growth, repair of body tissues, and asexual reproduction,
essential that the cells are identical so that the daughter cells would continue to code for essential proteins that can function properly

How to Get Seedless Watermelon?

Double the diploid number (2n) in a normal watermelon plant by the use of the chemical colchicine to tetraploid (4n).
Colchicine’s function?
Tetraploid’s gamete = 2n
Normal watermelon’s gamete = n
Fuse the tetraploid’s gamete (2n) with normal water melon’s gamete (n)

Why is triploid watermelon sterile?
During meiosis the normal pairing of chromosomes cannot properly take place since one set will have no homologous set to pair with

PROPHASE II
A spindle apparatus forms.
Chromosomes composed of two chromatids associated at the centromere
METAPHASE II
Chromosomes are positioned on the metaphase plate as in mitosis.
Crossing over in meiosis 
Two sister chromatids of each chromosome are not genetically identical
ANAPHASE II
Centromeres divide or replicate to allow separation of sister chromatids;
The chromatids move toward opposite poles as individual chromosomes.
ANAPHASE II
Add:
Direction of chromosome movement in Anaphase I at right angles to that in Anaphase II.
TELOPHASE II AND CYTOKINESIS
Nuclei form
Chromosomes begin decondensing
Cytokinesis occurs
4 daughter haploid cells
Each genetically distinct from the other
It’s Video Time!

Plant Cloning (Micropropagation)
And Revision
Tissue Culture
Vegetative propagation:
Stem, leaf or root cuttings
Micropropagation (tissue culture)
What are needed in micropropagation?
Tissue culture nutrient medium
(1) essential elements, or mineral ions, supplied as a complex mixture of salts;
(2) an organic supplement supplying vitamins and/or amino acids; and
(3) a source of fixed carbon: sucrose.
2. Plant growth regulators (Plant hormones)
Auxin
Cytokinin
3. Carefully controlled environment
Aseptic environment
No bacteria, fungus, virus, etc.

Step 1: Surface sterilization of explants
Small pieces of tissue (explants) removed from any part of the plant
May be meristematic (such as the shoot tip, the root tip), or non-meristematic (leaf parts).
First surface sterilized
Transferred in sterile surroundings to a sterile medium

Step 2: Callus formation (induction)
A callus is a mass of undifferentiated cell mass formed by mitosis

During callus formation, there is dedifferentiation
Consequence  lose the ability to photosynthesise.
Plant tissue culture cells have a small vacuole, lack chloroplasts and photosynthetic pathways and the structural or chemical features
Callus can later be induced to re-differentiate into whole plants by alterations to the growth media
Step 3: Subculturing
Aim:
callus can be subdivided to increase the number of plants eventually produced
Step 4: Shoots and roots formation
Auxin to cytokinin ratio
Step 5: Acclimatization followed by transplanting
Acclimatization allows environmental and physiological adaptation of tissue cultured plants to a greenhouse or a field environment
Before transplanting the plantlets

Advantages
extremely high multiplication rates.
identical copies of plants with desirable traits
Both seeds and pollinators are not required.
Plant cells can be genetically modified.
Limitations
aseptic condition requirements
A virus infection would quickly destroy a batch of plants
Suitable techniques of micropropagation are not available for many valuable species
Somaclonal variation may arise during in vitro culture
Revision/ Clarification
Tutorial: Enzymes
The initial rate of a reaction catalysed by an enzyme was measured at various substrate concentrations. Which graph shows the effect of a low concentration of non-competitive inhibitor on the reaction?

Topic: Isolating, Cloning and Sequencing DNA

Practical: DNA fingerprinting

Activity:          To establish identity of confiscated alligator meat.                

	Key procedure:             DNA fingerprinting Key words:                   VNTRs, restriction digestion

 

Skill A – Planning

An alligator is a reptile in the genus Alligator of the family Alligatoridae.  There are two extant alligator species: (i) the American alligator (Alligator mississippiensis); and (ii) the Chinese alligator (Alligator sinensis).  The Chinese alligator currently is found only in the Yangtze River valley and is extremely endangered, with only a few dozen believed to be left in the wild. Indeed, far more Chinese alligators live in zoos around the world than can be found in the wild!  Hence, the Chinese alligator is listed as a CITES Appendix I species, which puts extreme restrictions on its trade throughout the world. 

A consignment of suspected Chinese alligator meat, which is prized for its medicinal properties, has been confiscated by the Agri-Food and Veterinary Authority (AVA).  As its Chief Scientist, you have been tasked to plan, but not carry out, an investigation to verify if the meat originated from a poached Chinese alligator.

Your planning must be based on the assumption that you have been provided with the following equipment and materials which you must use:

·      muscle tissues from (i) confiscated consignment; (ii) an American alligator; (iii) a Chinese alligator

·      laboratory blender

·      DNA extraction buffer solution

·      micropipettors

·      microcentrifuge tubes

·      centrifuge

·      restriction enzyme

·      agarose gel

·      power pack, i.e. suitable source of current

·      nitrocellulose membrane

·      radioactive probe

·      autoradiography equipment

Your plan should: have a clear and helpful structure to include

·      an explanation of theory to support your practical procedure

·      a description of the method used, including the scientific reasoning behind the method

·      the type of data generated by the experiment

·      how the results will be analysed including how the origin of the organism can be determined

Pre-Task Survey

SPA Planning tasks that are designed around Application Syllabus topics might not follow the usual style as those designed around Core Syllabus topics.  Read the task given and complete the following (i) self-assessment; and (ii) flow chart.  Complete and submit them to your tutor’s pigeonhole by Tuesday, 17 August.

Self Assessment

Attempt this immediately, i.e. before you proceed to the next section.  Do take time to complete this so that your tutors can customise their lessons to aid your understanding.  

Criteria	My Comfort Level with Application Topics Planning Task*
	Comfortable	Somewhat comfortable	Uncomfortable	Remarks, i.e. What are some difficulties that I face? Why so?
1.     Providing theoretical basis for my suggested procedures
2.     Identifying what I want to observe / measure
3.     Identifying what I want to conclude
4.     Putting together the methods / procedure so that I can make my observations

* Put a tick in the box correlating to your comfort level

Draft I

Follow the guidelines given to come up with the first draft – this should be the mental picture that you should form within 1 min of analysing the exam question

Guidelines:

1.   An explanation of theory to support your practical procedure - theoretical consideration or rationale of the plan to justify the practical procedure

2.   A description of the method used including the scientific reasoning behind the method

I PAUSE

1. Identify the key procedure.  You might have many different sub-procedures / steps, some of which merely supports the key procedure.  The trick lies in the identification of the key.

Hint:     Scrutinise the aim of experiment. 

The command word in the aim is to establish identity

            What is one key procedure that relates to the command word?  Jot this down!

       DNA fingerprinting through RFLP analysis of VNTRs

2. Justify the procedure / method, i.e. what’s the scientific basis?

Hint:     Think about three main theoretical concepts surround the procedure that you are proposing.  Jot down the key words.

1.             VNTRs are DNA sequences, which are repeated in tandem a variable number of times at certain loci

2.            Restriction enzymes recognise and cut sites flanking the VNTR regions, producing restriction fragments of different lengths

3.            Alligator species exhibit tandem repeat polymorphism, hence each alligator species can be identified through the unique pattern of restriction fragments

3.   The type of data generated by the experiment

I PAUSE

3. Based procedure / method suggested, identify the observation / measurement that you can make.  How will these help you achieve the aim?

1.         Number and length of restriction fragments from each muscle tissue

2.         Assumption:

Confiscated consignment should have the same pattern of restriction fragments as that of the Chinese alligator, i.e. same number of restriction fragments, same length for each type of fragment

4. Identify two further sub-procedures / steps that you need to take to make the data meaningful.   

0.  Homogenise muscle tissues using ice-cold DNA extraction buffer in a blender

1.  Agarose gel electrophoresis to separate restriction fragments based on length

2.  Probe with complementary sequence to identify fragments bearing the VNTRs

                                                                                                   

4.   How the results will be analysed including how the identity of the organism can be determined

·           Compare restriction fragments obtained from (i) confiscated consignment; (ii) American alligator; (iii) Chinese alligator; 

·           Identify the restriction fragments that confiscated consignment has in common with each of the two species;

·           Determine the number of the restriction fragments that confiscated consignment has in common with each of the two species;

5.   The correct use of technical and scientific terms

I PAUSE

5. In a nutshell, identify the supporting sub-procedures / steps as instructed in d à draw a flow chart of critical steps.  You may want to consider the following details later for the actual write up:

·         how would you prepare the materials based on what you have been given? why do you select these conditions?

·         what are the steps involved in each procedure?  what is the scientific basis behind each step?

·         what observations / measurements would you want to take? 

·         how will these observations / measurements help you in identification?

Basic Plan of the Experiment (Answer parts a to e in a flow chart)

Homogenisation:  using ice-cold DNA extraction buffer and a blender

What does the ice-cold DNA extraction buffer contain?  Why must you use this buffer?

       ò

Centrifuge and transfer supernatant to a clean Eppendorf tube.  Add ice-cold ethanol. Centrifuge again and keep the pellet.

Why do you need to centrifuge to obtain supernatant?  Why do you need to ice-cold ethanol?

ò

Resuspend pellet in buffer.   Add restriction enzyme and incubate

ò

Agarose gel electrophoresis

What’s the basis of using AGE in relation to restriction digestion? 

ò

Southern blot, probing & autoradiography

What’s the basis behind these steps?