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?

Applications Topic: Isolating, Cloning and Sequencing DNA
Section B: Free Response Questions
1. Discuss
a) the aims of the human genome project. [6]
• to map all human genetic markers, i.e. by identification of their specific chromosomal location
• to construct a detailed physical map of the entire human genome
• to determine the base sequence of all 24 human chromosomes
• to develop technology for the management of human genome information
• to serve as an umbrella for similar mapping and sequencing projects on the genomes of other organisms, e.g. E.coli, yeast

b) the benefits of the human genome project. [8]
• improved diagnosis of disease and predisoposition to disease by genetic testing
• better identification of disease carriers through genetic testing
• better drugs can be designed using knowledge of protein structure
• greater possibility of correcting genetic disorders using gene therapy
• greater knowledge of family relationships through genetic testing, e.g. paternity testing in family courts
• advances forensic science through analysis of DNA at crime scenes
• improved knowledge of relationships between human and other organisms, which will help to develop better, more accurate classification systems.

c) the ethical concerns that have arisen about the human genome project. [6]
• unclear whether third parties should have rights to genetic test results – legislation is needed to ensure that there is no discrimination on the basis of genetic information
• reproductive issues regarding use of genetic information in reproductive decision making and reproductive rights. Prenatal genetic testing could lead to genetic manipulation or a decision to abort based on undesirable traits disclosed by the tests.
• Treatment vs enhancement of humans – no clear distinction between medical treatment and enhancement
• Fairness in access to advanced genome technologies – difficult to ascertain who should benefit or it will results in major worldwide inequities
• Commercialisation of products – difficult to determine who own genes and other pieces of DNA and to ascertain if patenting DNA sequences will limit the accessibility and development into useful products. [N08/P3/Q4]

2. a) Distinguish between a genomic DNA library and a cDNA library [6]

Feature Genomic DNA library cDNA library
DNA fragment cloning of all DNA fragments representing the entire genome, including coding and non-coding regions contains DNA fragments representing only the coding region of a genome.
Introns both the introns and the non - transcribed DNA are included in the clones intron sequences have been removed by RNA splicing during the formation of the mRNA, and a continuous coding sequence is therefore present in each clone.
Method DNA is cut using RE 
DNA fragments are incorporated into vector mRNA is extracted from a particular cells then reverse transcribe to form complementary DNA before incorporated into vector
Frequency All genes are represented equally in the library Genes that are transcribed abundantly will be represented more frequently in the library
No. of clones Larger number of clones to screen Smaller no. of clones to screen (as it contains only the coding regions)
Regulatory sequences Presence of regulatory sequences Absence of regulatory sequences


b) Describe the properties of plasmids that allow them to be used as cloning vectors. [6]
• Plasmids are circular, double-stranded DNA (dsDNA) molecules.
a) small molecules, size: a few kb to more than 100 kb
 facilitates the vector’s entry into host cells and the biochemical manipulation of DNA.
b) contain an origin of replication.
 The vector can replicate itself and the inserted gene of interest.
 During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell.
c) possess several unique restriction sites.
 The vector can be cut to insert the gene of interest.
d) contain genetic/ selectable markers.
 These are often resistance gene, which confer some well-defined phenotypes on the host organism which enables selection.
 i.e. allow us to identify the host cells that has taken up the recombinant DNA molecule.

c) Outline the large-scale production of a named important protein by genetic engineering.
[8]
• DNA sequences encoding the A and B chains of human insulin were chemically synthesised
• Each gene were placed under the control of the strong lac promoter and a part of the -galactosidase structural gene in the plasmid
• Both recombinanat plasmids containing the two artificial genes were transformed separately into E.coli.
• The 2 artificial genes were expressed independently as fusion proteins, consisting of the first few amino acid of -galactosidase  for initiation of translation in bacteria
• Lactose is added to induce transcription from the lac promoter
• Insulin fragment and -galactosidase were separated by a methionine residue
• Insulin polypeptides could be cleaved from the -galactosidase fragment by treatment with cyanogens bromide
• Purified A and B chains were attached to each other by disulphide bond formation
[N09/P3/Q4]