SPA Isolating, Cloning and Sequencing DNA

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?