Download Aimhigher Monitoring Template

Aimhigher Norfolk Monitoring and Evaluation Template
This template is provided to all projects funded by Aimhigher Norfolk to enable
consistent reporting on monitoring and evaluation across the county. The report
should be submitted by email to the Lorraine Sturman, Aimhigher Norfolk Manager –
Name of Project/Activity Raising Aspirations in Rural Norfolk
Report submitted by (Name) Dr Kay Yeoman
Contact telephone number 01603 592846 email [email protected]
For all sections, please provide only brief descriptive details. The impact monitoring
(question 2) should be the main emphasis of the report.
1
Executive Summary
Please use this page to give a brief overview of the project/activities using the following
headings.
a)
Progress since last year (for projects with continued funding from 2004-5)
This funding enabled us to continue the programme of raising aspirations in rural
Norfolk schools. We also expanded the programme to include 6th form workshops on
DNA transformation and PCR.
b)
Key achievements against objectives
The central aims of the project were firstly to raise the aspirations of pupils in rural
Norfolk schools, showing them that further and higher education is a possible and
achievable goal. Secondly, to widen the participation and understanding of science
for all age groups and abilities. The objective of the proposal was to create and
deliver exciting and inspiring science activities in the schools targeted. The project
achieved its objective. The activities were expanded upon from 2006.
c)
Challenges
Our challenges were to deliver science to a mixed ability audience and to try and engage
them all at some level with the activities on offer. We were also trying to introduce the
pupils to a world of higher education outside their sphere of experience and to encourage
them that they had the potential to achieve. The new challenge for this year was to
engage 6th form students in undergraduate-level practical classes.
d)
Proposed changes for next year
We would like to continue with the programme, but with a reduced number of schools.
We are continuing to do work with 6th from pupils. The equipment purchased over the last
two years will also be used in the Aimhigher funded project to raise the attainment level
of AS and A2 level biology.
2
1.
Activity Monitoring
Project leaders are asked to try to use the typology in the table below, as appropriate to each project - please refer to Annex A for examples against each
theme. It is recognised that activities are often designed to meet more than one aim and therefore could fit into more than one theme. Please select the
theme of best fit. If there are activities which can not be fitted into any other theme, then you may add an extra theme (Other) to suit local circumstances and
priorities. We appreciate that this is not an exact science but this standardisation of reporting will enable funders to have a better understanding of rough
volume of activities in each of these seven themes. We are not expecting detailed analysis of the volume so much as an indication of the volume and
balance of the different types of activity.
Theme (brief description of types of activities)
Volume (e.g. participants,
number of schools, colleges)

Awareness and aspiration raising activities
8 schools reaching 480 pupils

Attainment raising activities
8 schools reaching 480 pupils

Information, advice and guidance activities
8 schools reaching 480 pupils
Target group/s
Majority year 9 and 10, some year 8, 6th
form.
Majority year 9 and 10, some year 8, 6th
form.
Majority year 9 and 10, some year 8, 6th
form.
3
2.
Impact monitoring
In the evaluation pupils were asked if they had considered going onto higher education
before taking part in the event. They were then asked, as a result of the day, if they now
thought of going to university or college. The results are presented in Table 1.
Out of the eight schools visited, four returned the evaluation forms. There were 166
individual pupil returns, which represents approximately 35% of the target audience. We
were again disappointed that not every school returned the evaluation forms, despite
stressing the importance of evaluation of these types of project. As with the previous year,
even with the schools who did return their forms, it was clear from the number that not
every pupil who took part returned a form. We again tried to make the evaluation return as
easy as possible by providing a paid return envelope, but for some schools they clearly had
difficulty in reaching the pupils who had taken part. The suspected reason for this has not
changed, the end of the school year, when the project took place is a complex time for
schools, with pupils leaving after GCSEs and classes changing. It was not surprising that
teachers found it difficult to find the time to enable the pupils to complete forms. There is
still no easy solution to this issue. We prefer the pupils to fill in their evaluation a few days
after the event to enable self reflection and to see what aspects of the learning stood out in
their memories.
From the data presented in 2006, 48.5% of the pupils were serious about going into further
and higher education before and after the event. This year this increased to 58%, a jump of
10%. Although we had fewer returns this year, we feel that this may represent the
sustained efforts of Aimhigher to encourage thinking about participation in further and
higher education. It could also reflect the different group of schools which was visited this
year, although we think this unlikely as the schools were all rural, with a similar
performance profile. As with 2006, only 10% had not considered it before the event and
were still not interested afterwards. In 2007 19.8% were now going to seriously think about
going into higher education as a result of the activities (a 7% drop form the previous year).
This drop may reflect the increased number of pupils who were already serious about
entering higher education. In 2006, 8.6% were now definitely going to go after having
previously only though about it, this percentage stayed the same in 2007.
We feel that over the two years this project has been in place, we feel that we have
encouraged a substantial number of pupils to consider higher or further education.
Pupils’ engagement with the activities and the information learnt
In 2006 the pupil evaluation showed that the majority of the pupils enjoyed the DNA
sessions the most and the plants the least. This was not an unsurprising result as, in our
opinion, it is easier for the pupils to connect and engage immediately in this type of
practical work. This year the plant activity changed to an evolutionary activity with both
plants and animals. The pupils found the starfish particularly interesting and really did
remember lots of interesting details about the life and anatomy of this animal (as shown in
table 2a).
As with 2006, the facts that the pupils remembered this year were extremely encouraging,
considering that several days elapsed between the event and the evaluation.
4
Organisation and length of event
Each session offered to the schools lasted 45 -50mins. In some schools the sessions were
in direct succession, in others, one was before lunch and the other after lunch. Pupils were
asked their opinion on the length of the event; 74.3% thought that the event was just right
in length, 16.9% thought it too short and only 8.8% thought it too long. This data was
similar to that collected in 2006. The teachers felt that they had all received adequate
information about the event before it took place and gave the organisation a rating of 4.5.
We did arrive with little time to set up in some cases. This was due to the increased length
of time it took to prepare the animals at UEA for travel.
Medium term impact of the learning
One of the activities involved extracting DNA from their cheek cells. The DNA was
precipitated using ethanol and placed into a glass vial which could be worn as a necklace.
The pupils were allowed to take this memento home. During the activity we talked about
where DNA could be found, how it is inherited and the different reasons why you would
want to isolate and study DNA. The pupils in all schools engaged very well with this
question and answer session. The majority knew what DNA did, although few were able to
name it. Many of them knew that DNA was contained in the nucleus of most cells in the
body and some could even name the cells where it is absent (red blood cells). With the aid
of a model we talked about how DNA could replicate inside cells. Many of the pupils were
able to come up with good reasons why you would want to study DNA. Many of them had
heard of cloning and all of them know about Dolly the sheep but, in my opinion, fewer than
expected had heard of genetically modified organisms. Many of them knew about the
importance of DNA to forensics science and some were even able to talk about the use of
DNA to diagnose or cure disease. One pupil was even able to talk about the importance of
DNA to determine evolutionary relationships. During this 50 minute activity session, we
were able to re-enforce the importance of DNA as the blue print of life, engage the pupils in
its use and extend their knowledge of its structure and replication. This year, I was also
able to relate this DNA activity to the evolution activity; this was very powerful as it enabled
to pupils to appreciate how DNA can give rise to very different morphologies.
The second activity involved looking at animal and plant evolution. Using a range of live
specimens including starfish, earthworms, Hydra, water bears and a fascinating selection
of plants, including tree ferns and orchids, pupils were able to use their observation and
knowledge to place each organism in an evolutionary tree. During this 50 minute session
pupils were able to observe and engage with different plants and animals, using the naked
eye and microscopic analysis. Pupils had to look for links between the organisms that
might suggest they were related. They were encouraged to think about what made a plant
a plant, and an animal an animal, because with some of the specimens it was not obvious
which they were. Many pupils really engaged with the task, asking many questions and
really trying to work out the order of evolution, others just wanted to be told the answers.
The activity ended with a discussion on where these organisms should be placed in the
evolutionary tree. Pupils learnt about the plants’ move away from water, and that our
closest invertebrate relative was the starfish.
5
6th Form Workshops
In this two day workshop, pupils transformed the plasmid pGLO, containing the fluorescent
green protein from jelly fish into a bacterium Escherichia coli. The pupils were able to
induce the gene using the sugar arabinose. During this activity the pupils were taught about
plasmids and how they transfer and also their importance in the biotechnology industry.
In a second activity pupils extracted their DNA from cheek cells, and in a PCR reaction,
they were able to amplify a segment of their DNA. This DNA was analysed using DNA
electrophoresis. The produced booklet for the 6th form workshop is supplied in the
appendix.
6
Table 1 Impact of the project on pupils’ aspirations to enter further education
School
Before I was
seriously
thinking of
going. Now I am
still serious
about going
Before I had
thought about it.
Now I definitely
want to go
Before I had given it some
thought or I wasn’t sure. Now,
I am going to give it more
thought
Before I was
seriously thinking
of going. Now I am
going to give it
more thought
Before I hadn’t given it
much thought or I wasn’t
sure. Now I’m still not
going to give it much
thought, or I am not
sure.
Oriel
23
1
5
2
3
Hammonds
22
7
12
3
7
Archbishop Sancroft
24
2
5
0
1
Cromer
25
4
10
1
5
Total
94
14
32
6
16
Total % distribution
58%
8.6%
19.8%
There were 162 returns
There were 4 spoilt returns
7
3.7%
9.9%
Table 2 (a) The top three new things I learnt
Top three new things you learnt
Top three new things you learnt

Learning to handle starfish

DNA looks white

Starfish are hermaphrodite


How long ago plants evolved
Starfish have eyes and
thousands of legs

How to make my DNA


How to find and see DNA
Ethanol makes your DNA
show up

What DNA stands for


Some plants looked like animals and
some animals look like plants
Out of all the animals in the
room, the starfish was the
closest t to a human

Plants came before animals

Grass and plants with flowers aren’t that
old

Where the DNA in an animal is

Cells were the first things

What DNA looks like

How long ago plants and animals
evolved

About genes

DNA looks abit like a ladder

DNA can be found

Using a pipette

About water bears

Using proper microscopes

That worms and snails are linked


That you can make rabbits glow
That Dolly the sheep was
cloned

Starfish can push their stomach out of
their mouth

DNA is supercoiled

The chromosomes in our body

How DNA is built

What the nucleus was

Animals used not to have heads

We are all 99.9% the same

To consider all factors in evolution


Fish are vertebrates
Science is more fun than I
thought

Plants are green because of chlorophyll

DNA is a spiral

We have 23 pairs of chromosomes

You have cheek cells
Table 2 (b) What the pupils found most interesting and what they would like to know more
about
Most Interesting
I would like to learn more about

About DNA

Starfish

Plants and animals

Evolution

Starfish

Animals

DNA

Evolution of the
world
8

Microscopes
This year the pupils really enjoyed seeing and learning about the starfish, the majority of
them put a starfish related fact as one of the top three things they had learnt.
Teacher Assessment of the project
The teachers were asked to assess aspects of the event using a Likert-type scale with 5
points from poor (1) to excellent (5). The results are presented in table 3.
Table 3 Teacher rating of the event and separate activities (the 2006 data is in brackets)
Question
Rating
How would you rate this event as something that
might raise the aspirations of some of your pupils?
4.5 (4.63)
How would you rate the level at which the pupils
engaged with the activities?
DNA
Evolution
4.5 (4.8)
4.0 (4.0)
As can be seen from table 3, the teacher rating of the activities was very similar to the
previous year. All of the teachers felt that the pupils engaged well with all the activities, but
as with the previous year, that they probably liked the DNA activity the best, because as
one teacher stated “they felt like real scientists”. We supplied the pupils with white
laboratory coats for the DNA activity and this act of ‘dressing’ up made them feel quite
special. The impact that the coats had on the enthusiasm of their pupils was noted by one
of the teachers at Oriel High School and they are now seriously considering buying a stock
of coats for them to wear during science practicals. It was mentioned in one school that the
pupils would really like to have touched the animals. We were however reluctant due the
stress the animals had been under during travelling. One teacher also stated that “the
pupils were challenged to look at organisms in a way they hadn’t done before”. When
asked if an event at school or an event at UEA would be more helpful in raising aspirations,
100% of the teachers felt that the events in school were the most helpful, although three
teachers felt that ability for pupils to experience both would be beneficial. We also gathered
more data about what other activities would help raise aspirations and what in the teachers’
opinion were the main barriers to continuing into further and higher education. The results
of 2006 are presented in table 4a and the data collected in 2007 is presented in table 4b.
Table 4a Further activities and barriers faced by pupils (data collected in 2006)
What other activities would be most useful to
help raise the aspirations of your pupils?
What do you feel are the main barriers
faced by pupils at your school to
continuing in higher education?

Practical sessions (chemistry, and
environmental sciences)

Low self esteem


Lack of confidence
More science themed events

Finance
9

Meeting ‘real’ scientists

Lack of information

Physics subjects


Medically related experience
No parental experience of Higher
Education

Control and measurement
experiments

Low motivation and aspiration

Anti-clever ethic

Practicals which are new and different

Disruptive pupils

Involvement of past pupils

Low parental aspiration

Bugs and animal shows

Lack of expectation by parents

Any practical work we can’t do in
school because of lack of
funds/equipment

Lack of role models

Distances to travel

Family history

Pupils perceptions of science and
scientists

Science is hard to relate to

Not enough time for individual
investigations


Former pupils coming back to be peer
tutors and mentors to describe
experiences on degree courses
Similar activities to those offered in
this project
Table 4b Further activities and barriers faced by pupils (data collected in 2007)
What other activities would be most useful
to help raise the aspirations of your
pupils?

What do you feel are the main barriers faced
by pupils at your school to continuing in higher
education?
Include more physics and
chemistry (mentioned twice)

Forensic science

Pupils meeting successful UEA
students

Not expected by their families

Not discussed at home

There are no role models

Lack of self belief

Make science more interesting

Low personal aspiration

Family Background
The results presented in tables 4a and 4b are interesting. A recurring issue over the last
two years is family background and low parental aspirations. It seems important that future
funding should aim to address this issue. The involvement of past pupils who had gone
onto further and higher education was mentioned several times, as was meeting successful
UEA students and practising scientists as a way to raise aspirations. We are incorporating
these elements into the Aimhigher funded partnership project between UEA and two high
schools aimed at raising the attainment level in ‘A’ level biology. Teachers also commented
in both 2006 and 2007 that more of this type of activity is required in the chemical and
10
physical sciences. Perhaps Aimhigher could consider a special funding round to specifically
address developing activities in this area which can be taken out to schools.
In 2006 of those pupils who were seriously thinking of entering further or higher education,
78% of them knew of a family member or close friend who had gone. Only 22% of the
pupils seriously thinking of entering higher education of the pupils didn’t know anyone who
had gone. The data collected this year was very similar; of those who were seriously
thinking of going to University, 80% had a family member or close friend who had gone,
and 20% didn’t know anyone who had gone. This suggests a strong correlation between
knowing someone who had entered higher education and the desire to go themselves. This
also points to how important it is to change the attitude of parents and family groups. It is
encouraging to note however that 62% of the pupils expressed a desire to continue
education, 38% were not sure or knew it wasn’t for them.
Teachers were asked if they would like to see the project done differently, which subjects
they would like more information on and if there were further comments. The results are
presented in tables 5 (a) and (b)
.
Table 5 (a) and (b) Further comments by teachers
If we were to come back to your school at a
future date is there anything you would like to
see done differently?


Not sure if the evolution workshop was a
good topic, it needs re-structuring. They
spent too much time looking down
microscopes. There was too much time
left over.*
Any further comments

Arrive earlier to set up the
equipment

They enjoyed it and we
enjoyed it
In the evolution activity there was little
bringing the subject together and
learning anything new.*
* these comments were made by Archbishop Sancroft School, where one teacher gave the
evolution workshop a 1 rating for satisfaction. This was certainly atypical of all the other
schools we visited. It is important to include these negative comments in our evaluation
report, as it shows that for some schools there needs to be further thought about what we
offer. It is hard for us to receive such poor feedback as we worked very hard to ensure that
our events went well. It is also worth noting that a crucial factor in pupils engaging with
each event was the enthusiasm shown by their teachers, and this was lacking in the
evolution activity at Archbishop Sancroft.
11
Table 5 (b)
Which activities would you like further information on?

Follow on activities for evolution, especially advice on how to care for the live
animals

Further DNA activities
Many of the teachers also indicated that they would like more of their pupils to have the
opportunity to experience the activity and that we should consider doing this for the whole
year group. This is a worthy ambition, but one which would require further funding and a
team of people to run the activities, as it would not be possible for us to cover this number
at this present time.
Publicity
This project has featured in the UEA Beacons for Communication application. There was
not as much interest from the press this year, but several local newspapers did cover the
events.
Now that the project has run for two years, it is anticipated that the results of this work be
published in a school educational journal.
Long term impact of the project
We feel that this type of activity should be maintained each year if possible in order to
maintain momentum.
Further reflection
There has been a substantial build up of equipment and resources which need to be
maximised. The equipment must be entered onto the Aimhigher website. I have already
lent the equipment to other schools, including primary schools running different types of
project, such as soil analysis. I have also used the equipment to run forensic science days
at Marshlands High School, Norwich School for boys and Avenue Middle School. More of
these forensic science days are being planned. We also used the equipment in the Norwich
Castle event “Poo, Puke and Pee”, which attracted over 1000 visitors. The equipment will
also be used in the Aimhigher funded project to raise the attainment level of AS and A2
level biology. UEA is willing to still store and maintain the equipment to ensure that it is safe
for pupils to use.
Conclusion
As with the previous year, we feel that the project has been successful. We were able to
visit very rural schools and give the pupils an interesting experience which may prove
pivotal in their decision to go into Higher Education.
6th form workshops
12
We had planned to do three 6th form workshops with Methwold, Rosemary Musker and
Hammonds. Unfortunately Methwold High school pulled out a few days before the event
and I am in the process of trying to re-arrange a time suitable for the school. We travelled
to Rosemary Musker and set up for the workshop to find only three out of the fifteen
registered pupils there, unfortunately no teacher was present and we felt that we could not
run the event without the presence of the teacher for health and safety reasons. The
Hammonds event took place on the 9th and 10th of July, there were 12 pupils and three
teachers present. The event was very successful, with the pupils learning new skills and
gaining confidence. One pupil was now considering doing biochemistry as a degree, as he
had enjoyed the practical so much.
Mr Stuart Wright, our contact at Hammonds said “The sixth formers were still buzzing about
your workshop even at the end of term - thanks for that!”
Dr Kay Yeoman will be doing the 6th form workshop with CNS on the 22nd November 2007.
13
6.
Financial Monitoring
Please amend the rows as necessary.
Main headings of expenditure as
identified in project proposal/service
level agreement.
Proposed
expenditure
of funds
identified in
Project Plan
(£)
Actual expenditure of funds
to 31 July 2007
(actual expenditure should
not include funds allocated
or committed but not
spent) (£)
Over or
underspend at
end of July
2007
(£)
Reasons for any
variation in spend
of 15% or more
Capital Equipment
4, 250
5, 039
+789
Consumables, including printing
2,000
2, 374
+374
500
680
+180
3000
2088.78
-911.22
The equipment was
more than
anticipated; we
reduced the staff
costs to
compensate.
The cost of the kits
from BioRad
increased this year.
Travelled further
than expected
To balance the
overspend on
equipment
See above
9, 750
10, 181.78
+431.78
Travel
Staff time
TOTAL
14
APPENDIX
6th Form Workshops
Dr Kay Yeoman
School of Biological Sciences
University of East Anglia
15
Transforming the Green Fluorescent Protein Gene from
Jellyfish into the bacterium Escherichia coli
Background
In this lab you will perform a procedure known as genetic transformation. Genetic
transformation literally means change caused by genes, and involves the insertion of a
gene into an organism in order to change the organism's trait. Genetic transformation is
used in many areas of biotechnology. In agriculture, genes coding for traits such as frost,
pest, or spoilage resistance can be genetically transformed into plants. In bioremediation,
bacteria can be genetically transformed with genes enabling them to digest oil spills. In
medicine, diseases caused by defective genes are beginning to be treated by gene
therapy; that is, by genetically transforming a sick person's cells with healthy copies of the
defective gene that causes the disease.
You will use a procedure to transform bacteria with a gene that codes for Green
Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish
Aequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and glow in
the dark. Following the transformation procedure, the bacteria express their newly acquired
jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant
green colour under ultraviolet light.
In this activity, you will learn about the process of moving genes from one organism to
another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally
contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually
contains genes for one or more traits that may be beneficial to bacterial survival. In nature,
bacteria can transfer plasmids back and forth allowing them to share these beneficial
genes. This natural mechanism allows bacteria to adapt to new environments. The recent
occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids.
Rio-Rad's unique pGLO plasmid encodes the gene for GFP and a gene for resistance to
the antibiotic ampicillin. pGLO also incorporates a special gene regulation system, which
can be used to control expression of the fluorescent protein in transformed cells. The gene
for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells'
nutrient medium. Selection for cells that have been transformed with pGLO DNA is
accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type
phenotype) on plates not containing arabinose, and fluorescent green when arabinose is
included in the nutrient agar medium.
Your task will be:
1. To do the genetic transformation.
2. To determine the degree of success in your efforts to genetically alter an organism.
16
Procedure
1) In your tray you will find two small plastic eppendorf tubes, with your pen label one
+pGLO and the other –pGLO. Write your initials on these tubes.
2) Take the tube from your tray labelled ‘TM’, this contains transformation mix. Add
250 µl of transformation mix to each of your tubes labelled +pGLO and –pGLO.
3) Place these tubes in your cup of ice.
4) In your tray you will find a starter plate containing the bacterium E. coli, HB101.
Take one of your sterile loops and pick off a colony of the E. coli. Pick up the
eppendorf tube labelled +pGLO and immerse the loop in the transformation mix.
Spin the loop between your index finger and thumb until the entire colony has
dispersed in the transformation mix. Place this tube back into the ice cup and
repeat this procedure for the tube labelled –pGLO.
5) Take the 10 µl fixed volume pipette and place a yellow tip on the end. In your tray
you will find a blue eppendorf tube labelled plasmid. Take out 10µl of the pGLO
plasmid and add it to the tube labelled +pGLO (which is in your ice cup).
6) Question Why do you not add the plasmid to the –pGLO tube?
7) Incubate the tubes on ice for 10 minutes. Make sure the liquid in the tubes is in
contact with the ice.
8) While the tubes are on ice you can label your plates. You have four agar plates.
Write your names on the bottom all of the plates and then label them according to
the table below.
Plate with label
Write this on the plate
LB
-pGLO
LB amp
-pGLO
LB amp
+pGLO
LB amp/arabinose
+ pGLO
9) When your tubes have finished their incubation on the ice, then place them in the
foam rack and put them in the 42oC water bath for 1 minute. This is called heat
shocking.
10) Then place your tubes immediately back in the ice cup and leave for 2 minutes.
11) In your tray you will find a glass bottle labelled LB. Take out 200 µl of the liquid and
add it to your tube labelled +pGLO. Repeat for –pGLO. Incubate both tubes at room
temperature for 10 minutes.
12) Take your plate labelled LB –pGLO. Take your fixed volume 100 µl pipette and add
a yellow tip on the end. Take out 100 µl of the tube labelled –pGLO, take the lid of
the plate and squirt the liquid onto the surface of the plate. Take a clean loop and
spread the liquid evenly across the surface of the plate, by quickly skating the flat
surface of the loop across the surface of the plate.
17
13) When your plates have dried. Stack them with the lids downwards and tape them
together with masking tape. These will be incubated at 37oC overnight.
Next Day
Review Questions
1) On which of the plates would you expect to find bacteria most like the original
untransfomed E. coli colonies you used?
2) If there are any genetically transformed bacterial cells, on which plate(s) would you
expect to find them?
3) Which plates should be compared to determine if any genetic transformation has
occurred?
4) What is meant by the control plate? What purpose does this control plate serve?
18
Data Collection and Analysis
1) How much bacterial growth do you see on each plate? Record your observations in
the table below.
2) If you can, count the number of colonies on each plate and record your
observations in the table below.
3) Using your UV light pen and or the UV light box, observe each of your plates. Do
any of the bacterial colonies glow green? Record your observations in the table.
Plate
Amount of bacterial
growth
Colony
count
Do the bacterial colonies
fluoresce?
+pGLO, LB/amp
+pGLO, LB
amp/arabinose
-pGLO, LB/amp
-pGLO, LB
What conclusions can be drawn from the observations above?
19
Investigating your genes using PCR
Background
Genes and DNA
It is estimated that the 23 pairs of chromosomes (46 total chromosomes) of the human
genome contain a total of 30,000–50,000 genes. Each gene holds the code for a particular
protein. Interestingly, these 30,000–50,000 genes comprise only about 5% of chromosomal
DNA. The other 95% is noncoding DNA. This noncoding DNA is found not only between,
but within genes, splitting them into segments. In eukaryotes, these sequences within
genes (called introns) are transcribed into RNA but in the end do not make a protein called
introns. The sequences that do code for proteins are called exons. Both introns and exons
are initially transcribed, then introns are spliced out of the RNA to create messenger RNA
(mRNA).
In eukaryotes, genomic DNA is transcribed into RNA molecules containing both introns and
exons for a particular gene. While the RNA is still in the nucleus (before being transported
out of the nucleus), the introns (in = stay within the nucleus) must be removed from the
RNA while the exons (ex = exit the nucleus) are spliced together to form the complete
coding sequence for the protein. This process is called RNA splicing. Some genes may
contain a few introns, others may contain dozens.
Throughout evolution, intron sequences have been the target of random insertions by
short repetitive interspersed elements, also known as SINEs. SINEs have become
randomly inserted within our introns over millions of years. One such repetitive element is
called the Alu sequence. This is a DNA sequence about 300 base pairs long that is
repeated, one copy at a time, almost 500,000 times within the human genome. The origin
and function of such randomly repeated sequences is not yet known. The Alu name comes
from the AluI restriction enzyme recognition site that is found in this sequence.
In this activity, you will isolate your own genomic DNA from your cells. You will use primers
that flank both the entire Alu insertion (300 base pairs in length) and 641 base pairs of the
PV92 locus to amplify a 941 base pair fragment (if the Alu element is present) or a 641
base pair fragment (if the Alu element is absent). Agarose gel electrophoresis of the PCR
products is sufficient to distinguish among homozygotes (+/+) for the presence of the Alu
repeat (941 base pair product only), homozygotes (–/–) for the absence of the Alu repeat
(641 base pair product only), and heterozygotes (+/–) having both the 641 and the 941
base pair products.
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Procedure
1) Get a clean eppendorf tube from your tray and label it with your initials.
2) Obtain a cup of saline solution. Gently chew the inside of your cheeks for 30
seconds. Place the saline in your mouth and rinse vigorously for 30 seconds. Expel
the saline back into the cup.
3) Transfer 1ml of the saline rinse into the eppendorf with your initials and spin the
tube for 2 minutes at full speed in the centrifuge.
4) After centrifugation, you should see a white pellet of your cheek cells. Remove the
saline, by pouring it off; be careful not to loose your pellet!
5) Resuspend the cell pellet by flicking the tube so that no clumps of cells remain.
6) Take the tube from your tray labelled IG, this contains the InstaGene matrix.
7) Using the fixed volume 20 µl pipette, with a yellow tip on the end, transfer all of your
resuspended cheek cells into the tube labelled IG. Screw the cap on tightly and
shake to mix the contents. Label this tube with your initials.
8) Place the tube at 56oC for 10 minutes, after 5 minutes shake them, then replace.
9) Remove your tube, shake and place it at 100oC for 5 minutes.
10) Allow the tube to cool, then centrifuge it for 5 minutes. The supernatant is your DNA
TEMPLATE.
11) Take one of the PCR tubes from your tray and a capless tube. Label the PCR tube
with your initials and place it inside the capless tube.
12) Transfer 20 µl of your DNA TEMPLATE into the PCR tube.
13) In your tray you will see a tube marked MM, this is the Master Mix. Transfer 20 µl of
the Master Mix into your PCR tube. Mix by pipetting up and down a few times.
14) Place your tube inside the PCR thermocycler machine, this will be run over night
and we will be able to analyse the PCR DNA products tomorrow.
Next Day
Make your agarose gel according to the instructions on the sheet, you will be given a
demonstration on how this is done.
1) Remove your tube from the PCR machine.
2) In your tray you will find a tube labelled LB, this is loading buffer and it is blue. Take
your fixed volume 10 µl pipette, with a yellow tip on the end and transfer 10 µl of the
blue dye into your PCR tube.
3) Load your sample into the well on the agarose DNA gel.
This will now run for a few hours.
Staining the agarose gel
1) Remove the electrodes from the powerpack and take out the gel tray (make sure
the gel doesn’t slip off!)
2) Gently slide the gel from the tray into the gel box.
3) Add 120ml of 100x Fast Blast stain into the tray containing the gels.
4) Stain the gels for 2 minutes with gentle agitation.
5) Pour off the stain into the beaker provided; the stain can be used again.
21
6) Transfer the gel into the sandwich box and rinse with warm tap water for approx 10
seconds.
7) Destain the gel by washing twice in warm tap water for 5 minutes each, with gentle
shaking.
8) Leave to allow the bands to develop.
Analysis of the Results
1) Compare your sample lanes with the control lanes using the DNA size marker as a
reference.
2) Mark the location and size of your fragment or fragments. By comparing your DNA
migration pattern to the controls, determine whether you are homozygous +/+, homozygous
–/–, or heterozygous +/–. If your sample lane is blank, discuss the possible reasons with
your classmates.
Remember that the interpretation of this gel allows you to determine your genetic makeup
only at the locus (chromosomal location), being studied. There are three possible
genotypes for the Alu insert at the location you have amplified. For a class, determine the
number of individuals of each genotype: homozygous +/+, homozygous –/–, and
heterozygous +/–. Tally the class results in the table below.
22
Review of Molecular Biology
This section provides an overview and concepts with which students should be familiar in
order to get the most out of this lab. Please also refer to the Glossary Section for definitions
of molecular biology terms.
Any living organism functions based on the complicated interactions among nucleic
acids, proteins, lipids (fat), and carbohydrates. In nearly all cases, certain proteins, termed
enzymes, control the almost infinite number of interactions and life processes in living
creatures. Think of enzymes and proteins as all the different people on earth. Each person
performs a different role, function, or job on this planet, and although people are not the
actual physical make-up of buildings, documents, food, and roads, it is the people that
make these buildings and roads, and write the documents, and plant and nurture the crops.
In the same way, enzymes and proteins do not comprise bones, lipids, sex hormones, and
sugars, but enzymes control these structures, their interactions, and processes. Because
proteins and enzymes ultimately play such a critical role in the life process, scientists have
spent many lifetimes studying proteins in an attempt to understand how they work and how
they can be controlled. With a complete understanding, we could cure, prevent, and
overcome many diseases and physical handicaps as well as explain exactly how and why
organisms exist, propagate, and die. However, the complete answers do not lie solely in
the knowledge of how enzymes function; we must learn how they are made.
Before we can control enzymes, we must understand where they come from and what is
the basis of the molecular information that encodes proteins. That answer lies within our
genetic code.
Each living organism has its own blueprint for life. This blueprint defines how an
organism will look and function (using enzymes as a means to form the appearance and
control the functions). The blueprint codes for all the different enzymes. With amazing
precision, this blueprint gets passed on from generation to generation of each species.The
transfer of this blueprint from generation to generation is called heredity. The blueprint for
any organism is called its genome. The hereditary code is encrypted within the sequence
of the DNA molecules that make up the genome. The molecule that constitutes the genome
and thus the hereditary code is DNA (deoxyribonucleic acid). The genome consists of
very long DNA/protein complexes called chromosomes. Prokaryotes, organisms lacking a
true nucleus, have only one chromosome. All other species, eukaryotes, have a defined
cell nucleus that contains multiple chromosomes. The nucleus is a defined, membraneenclosed region of the cell that contains the chromosomes. The number of chromosomes
varies with the organism — from 2 or 3 in some yeasts to up to 100 or so in some fish.
Humans have 46. In most cases, chromosomes come in nearly identical pairs (one
member of the chromosome pair from each parent). In general, the members of a pair differ
in small details from each other, since they come from different parents, but are otherwise
identical or homologous. Cells with homologous pairs of chromosomes are called diploid.
Nearly all cells of an organism are diploid. Cells that have only one chromosome of each
pair are called haploid. All sperm and ova are haploid.
The process of forming sperm and ova is called meiosis. Meiosis starts with a diploid
cell that divides into two haploid cells. When a sperm fertilizes an ovum, the two nuclei
fuse, and thus the new nucleus contains pairs of each chromosome, one partner from each
parent. The result is called a diploid zygote.
All cells of diploid organisms duplicate chromosomal pairs when they divide (except
23
when sperm and ova are formed), so that all body cells (called somatic cells) of an
organism are diploid. The process of cell division in which the chromosomes are duplicated
and each daughter cell gets pairs of chromosomes is called mitosis. It is through the
processes of mitosis and meiosis that the hereditary code is passed from cell to cell and
generation to generation. Now that we know where the code is and how that code is
passed on, we need to know how the code produces the enzymes that control life. The
actual DNA code for a protein is contained within a segment of a chromosome called a
gene. In nearly all cases, diploid organisms will have the same gene on a specific
chromosome pair. Each gene on a particular chromosome of a specific chromosome pair is
also called an allele.
To clarify, a gene encodes a particular protein that performs a particular function. An
allele is a specific version of a gene on a particular chromosome. Thus, there are genes for
hair colour and there is an allele for the hair colour gene on each chromosome pair. The
gene or allele’s DNA code can also be called the genotype.
When the protein is made from this code and performs its function, the physical trait or
result that is seen is called the phenotype. In many cases the two alleles on the specific
chromosome pair coding for a protein differ slightly in their respective DNA code
(genotype).
Any slight difference in code between the two alleles can result in two different proteins,
which, although intended to perform basically the same function, may carry out that
function slightly differently, causing different results and thus different phenotypes.
Therefore, it is not only the various combinations of chromosomes a parent contributes to
each offspring, but also the various combinations of alleles and how each of the enzymes
coded from the alleles work together that decide how we look and allow us to function. The
various combinations are nearly infinite and that is why we are all different.
The study of genotypes and phenotypes is often referred to as Mendelian genetics
(after Mendel, the individual who pioneered the study of heredity and genetics).
DNA: What Is It?
A DNA molecule is a long polymer consisting of four different components called
bases. The four bases are also called nucleotides. It is the various combinations of these
four bases or nucleotides that create a unique DNA code or sequence (also genotype,
gene, and allele). Nucleotides are comprised of three different components:
• Nitrogen base
• Deoxyribose sugar
• Phosphate group
Each nucleotide contains the same ribose sugar and the phosphate group. What
makes each nucleotide unique is its nitrogen base. There are four nitrogen bases:
Adenine (A)
Thymine (T)
Guanine (G)
Cytosine (C)
A DNA nucleotide chain is created by the connection of the phosphate group to the
ribose sugar of the next nucleotide. This connection creates the “backbone” of the DNA
24
molecule. To designate the different ends of this single-stranded chain, we use some
typical
biochemistry terminology, in which the carbons on any sugar are numbered. The sugar of a
nucleotide contains 5 carbons. The phosphate group (PO4) of a given nucleotide is
connected to the 5' carbon of the sugar. A hydroxyl group (OH) is attached to the 3' carbon
of the sugar, and this 3' OH group connects to the phosphate group of the next nucleotide
in the chain. Thus, the end of a single-strand DNA molecule that has a free phosphate
group (i.e., not attached to another nucleotide) is called the 5' end, and the end of the DNA
molecule (with no subsequent nucleotide attached) is called the 3' end.
It has become standard that a single-stranded DNA molecule is written with the 5' end
on the left and the 3' end on the right. Therefore, a single-stranded DNA chain’s sequence
is represented from left to right, starting on the left with the 5' nucleotide and moving to the
right until the 3' nucleotide is last. Most DNA sequences are read 5' to 3'. However, the long
DNA molecules or chains that comprise the chromosomes are not single-stranded
molecules. From X-ray crystallography patterns of DNA, and some imaginative molecular
model building, Watson and Crick deduced that DNA is in fact a double-stranded
molecule with the two single strands of DNA held together by hydrogen bonds between
the nitrogen bases (A, T, G, and C). This double-stranded molecule is often called a
duplex. There are several important properties of double-stranded DNA molecules.
• Chromosomal (also called genomic) DNA is double-stranded.
• The overall structure is that of a helix with two strands intertwined.
• The structure can be viewed as a twisted ladder.
• The phosphate-deoxyribose backbones are the sides of the ladder.
• The nitrogen bases (A, T, G, and C) hydrogen bonded to each other are the rungs.
• Only the nitrogen bases A and T and C and G can form hydrogen bonds to each other.
When A binds to T or C binds to G this is considered base pairing. Neither C and T, nor A
and G form hydrogen bonds.
• The two strands are antiparallel; that is, the strands are oriented in opposite directions.
This means that the ladder runs 5' to 3' in one direction for one strand and 5' to 3' in the
opposite direction for the other strand.
DNA Structure Conclusions
• Because A only binds to T, and G only binds to C, the two strands will have exactly the
opposite, or complementary, sequence running in opposite directions (one strand 5' to 3',
the other 3' to 5').
• These two complementary strands anneal or hybridize to each other through hydrogen
bonds between the bases.
• A new strand of DNA can be synthesized using its complementary strand as the
template for new synthesis.
• Each strand carries the potential to deliver and code for information.
The length of any double-stranded DNA molecule is given in terms of base pairs (bp). If a
DNA strand contains over a thousand base pairs, the unit of measure is kilobases
(1 kb = 1,000 bp). If there are over one million base pairs in a strand the unit of measure is
megabases (1 Mb = 1,000 kb).
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DNA Replication — Strand Synthesis
New strands are synthesized by enzymes called DNA polymerases. New strands are
always synthesized in the 5' to 3' direction. For a new single strand of DNA to be
synthesized, another single strand is necessary. The single strand of DNA that will be used
to synthesize its complementary strand is called the template strand.
However, in order for DNA polymerase to start synthesizing a new complementary
strand, a short stretch of nucleotides (approximately 20 base pairs long) called an
oligonucleotide primer must be present for the polymerase to start synthesis. This primer is
a short stand of nucleotides complementary to the template where the researcher wants
synthesis to begin. The primer must have a free 3' hydroxyl group (OH) for DNA
polymerase to attach the 5' phosphate group of the next nucleotide.
The DNA polymerase grabs free (single) nucleotides from the surrounding environment
and joins the 5' phosphate of the new nucleotide to the 3' hydroxyl group (OH) of the new
complementary strand. This 5' to 3' joining process creates the backbone of the new DNA
strand. The newly synthesized strand maintains its complementarity with the template
strand because the DNA polymerase only joins two nucleotides during new strand
synthesis if the new nucleotide has its complement on the template strand. For example,
the DNA polymerase will only join a G to the 3' end of the newly synthesized strand if there
is the C counterpart on the template strand to form a hydrogen bond. Guanine will not be
joined to the new strand if adenine, thymine, or guanine is the opposite nucleotide on the
template strand.
DNA polymerase and strand synthesis allow DNA to replicate during mitosis. Both new
DNA strands are synthesized simultaneously from the two original DNA template strands
during mitotic DNA replication.
As you can see, DNA, RNA, and proteins are closely tied to each other. Thus, you
can realize why researchers today, in an attempt to understand the mechanisms
behind the various life processes, must study the nucleic acids as well as the proteins to
get complete answers about the flow of information carried in the genetic code. In the last
20 years, many gains in the areas of nucleic acid techniques have finally allowed
researchers the means to study the roles of nucleic acids in life processes.
Individual discoveries by many scientists have contributed the pieces that have begun
to solve one of the most mysterious puzzles of life — understanding the hereditary code. In
1985, enough pieces of the puzzle were in place for a major breakthrough to occur. This
understanding of how the necessary molecular components interact to faithfully replicate
DNA within living cells led to the development of a technique for creating DNA in a test
tube. This technique is called the polymerase chain reaction, or PCR.
26
Glossary of Terms
Aliquot The division of a quantity of material into smaller, equal
parts
Allele A variation of a gene on a particular chromosome
Alu A small piece of repetitive DNA that contains the AluI
restriction enzyme site, from which the sequence
obtained its name
Annealing Binding of oligonucleotide primers to complementary
sequences on the template DNA strands
Biotechnology The manipulation of organisms (microbes, plants or
animals) DNA to help solve human problems
Chelate To bind metal ions in solution. An example of a common
chelating agent is EDTA or ethylenediamine tetraacetic
acid
Cofactors Ions or small molecules needed by an enzyme to
function properly. For example, TaqDNA polymerase
needs Mg2+ in order to function properly. Mg2+ would
therefore be considered a cofactor
Denature The process of melting apart two complementary DNA
strands. In vivo denaturation is accomplished by
enzymes; in the PCR reaction denaturation is
accomplished by heat
DNases Digestive enzymes that degrade DNA
dNTPs Commonly used abbreviation for all four deoxynucleotide
triphosphates (dATP, dTTP, dGTP, dCTP) used in
synthesizing DNA
Ethidium bromide A fluorescent dye molecule that intercalates between
DNA base pairs and fluoresces when exposed to
ultraviolet light
Eukaryotes Organisms that are made up of cells containing a
membrane-bound nucleus that contains the genetic
material (DNA)
Exon The region of a transcribed messenger RNA molecule
that gets spliced together and leaves the nucleus for
translation into protein sequence
Extension This refers to the process of Taqpolymerase adding
dNTPs (deoxynucleotide triphosphates — dATP, dTTP,
dCTP, or dGTP) onto the ends of the oligonucleotide
primers. Extension follows the base pairing rule and
proceeds in the 5' to 3' direction
Genome The sequence of DNA molecules within the nucleus that
codes for all proteins for a given species. Each segment
of DNA that encodes a given protein is called a gene. The
information contained in the genome constitutes the
organism’s hereditary code
27
Genomic DNA The sum total of the DNA that is found within the nucleus
of a cell
Genotype The combination of alleles carried by an individual
Hardy-Weinberg equilibrium The conditions that enable a population to maintain its
genetic frequencies; These conditions are: large
population, random mating, no immigration or emigration,
no mutations, and no natural selection
Homologous chromosomes A pair of complementary chromosomes that contain the
same genetic sequences, or genes, with one
chromosome inherited from the mother and one
chromosome inherited from the father
InstaGene matrix Microscopic beads that bind divalent cations in solution;
The binding or sequestering of divalent cations prevents
their availability to enzymes that can degrade the DNA
template
Intron The region of a transcribed messenger RNA that is
spliced out of the mRNA and is not translated into
protein sequence
Lysis The process of rupturing a cell to release its constituents. In
this exercise, human cheek cells are lysed to release
genomic DNA for PCR reactions
Master mix The main solution of a PCR reaction which contains all of
the necessary components (dNTPs, primer, buffer, salts,
polymerase, magnesium) of the reaction except the
template DNA
Messenger RNA A type of RNA that is synthesized from the genetic
material (DNA) and that attaches to ribosomes and is
translated into protein
Molecular biology The study of genes and the molecular details that regulate
the flow of genetic information from DNA to RNA to proteins,
and from generation to generation
Nucleotides The fundamental unit of DNA or RNA; they consist of a
sugar (deoxyribose or ribose), phosphate, and
nitrogenous base (adenine, thymine, cytosine, or
guanine, with uracil in place of thymine in RNA)
PCR Polymerase chain reaction. The process of amplifying or
synthesizing DNA within a test tube
Primer A short sequence of nucleotides (usually 16–24 bases in
length) that recognizes a particular sequence of
nucleotides on the target DNA sequence; primers for the
polymerase chain reaction are usually synthesized in a
laboratory
Reagents Materials needed to conduct an experiment; they are
usually solutions or mixtures of various solutions
Taq DNA polymerase Thermostable DNA polymerase that was isolated from the
thermophilic bacterium Thermus aquaticus. This DNA
28
polymerase is commonly used in PCR reactions
Template The strand of DNA that contains the target sequences of
the oligonucleotide primers and that will be copied into its
complementary strand
29