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Transcript
Life on Mars
Student Notes
Life on Mars?!
Welcome to the MARSLANDER laboratory. Today you will to be analysing samples
collected on Mars, looking for signs of life. We have been given samples from a number of
Martian locations:
1. A desert area in the northern hemisphere
2. The foot of an extinct volcano in the southern hemisphere
3. The floor of a large canyon system
4. A rocky equatorial region
5. The northern polar region.
6. The southern polar region.
We will be looking for DNA (deoxyribonucleic acid), the chemical that carries the
instructions for all life on Earth. Using a very powerful technique called PCR (polymerase
chain reaction) it is possible to amplify short sections of DNA to detectable levels, using
very small biological samples. Click here to see a short clip explaining how this done.
In addition to analysing the samples from Mars it is important to carry out control
experiments. A ‘positive control’ PCR, using a sample we know contains DNA is used to
check that the PCR is working. A ‘negative control’, without DNA, is carried out to check
that samples have not been contaminated during PCR preparation. Positive controls can
also be used to exclude so-called “false-positive” results.
Practical session – Laboratory
First you will practise using micro-pipettes and then you will run out the products of the
PCR reactions on a gel over the course of this session, as described below.
Agarose Gel Electrophoresis for Size-Separation of DNA
In order to identify the different sizes of DNA you will need use a process called agarose gel
electrophoresis. This process uses an electric current passed through a gel to separate
DNA fragments by size.
An agarose gel is made by heating powdered agarose (a
seaweed extract) and a buffer solution. This produces a thick liquid which is poured into a
sealed tray and allowed to set. The gel is then put into a buffer solution, and DNA
samples are put into special holes called “wells” that have been created in the gel. An
electric current is passed through the gel, and because the DNA fragments are negatively
charged, they are drawn towards the positive electrode. The smaller the DNA fragment,
the faster it travels along the gel. This results in DNA separation by size, with the smaller
fragments migrating further than the larger fragments on the gel. DNA fragments can be
visualised under UV lights.
Smallest-sized
DNA fragment
Negative
electrode (-)
Positive
electrode (+)
Wells for
sample
loading
Largest-sized
DNA fragment
Direction of travel of DNA fragments
You will be given your PCR products. There are 8 tubes (labelled 1- 8) and a
tube labelled “M”. Tube M is the DNA size marker which allows you to estimate the size of
your bands.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html
Your demonstrator will give you some loading dye. We have to add this to the DNA
samples in order to see where we are loading them on the gel, and the dye also makes
the samples heavy so they will sink to the bottom of the wells of the gel.
Add 2µl of loading dye to each sample. Carefully mix the dye in the sample by pipetting up
and down.
Load 10 µl of each sample in order i.e. DNA Marker first, followed by tubes 1-8. Your
demonstrator will show you how to load a sample on a gel. Once all the samples have
been loaded your demonstrator will then plug the gel in and allow it to run.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html
Practical Session – Computer Section
Below is an example of samples that we have previously examined from Mars. What is
your initial deduction from the results below?

DNA marker ladder (to estimate the size of the unknown samples)

negative control – to show that the PCR has worked without crosscontamination from other samples

positive control – human GAPDH gene. We use it to test that the PCR has
worked properly and helps to show us that we have excluded human
contamination in our samples

sample 1 - a desert area in the northern hemisphere

sample 2 - the foot of an extinct volcano in the southern hemisphere

sample 3 - the floor of a large canyon system

sample 4 - a rocky equatorial region

sample 5 - the northern polar region

sample 6 - the southern polar region
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html
Analysing the PCR products
What can we do next with the samples? The next step is to try and identify in more detail
what the samples may be. We have already analysed your PCR products by a process
called “DNA sequencing” in order to deduce the order of the individual bases in the PCR
product.
We sequenced the DNA band from samples 2 (foot of extinct volcano) and 4 (rocky
equatorial region), and we have got the sequences on the computers for you to analyse.
You will need to look at the Word files containing the DNA sequences of your PCR
products. For example, the sequence from Sample 4 is about 476 base pairs long and
the translated protein sequence deduced from this DNA sequence, is shown on page 9.
We are going to compare the sequence we have identified by copying and pasting the
sequence into a DNA sequence computer search engine called BLAST. The BLAST
programme allows you to input a DNA sequence of interest and BLAST will identify
regions of similarity between sequences that are stored on its DNA database. The
program compares nucleotide (or protein) sequences to sequence databases and
calculates the statistical significance of matches.
The results of the search identifies
genes that are found in organisms (e.g. human, mouse or bacterial), that are most closely
matched to your sequence. It will give us a percentage identity, i.e. how many of the input
sequence bases match bases from the gene in the matched organism.
Today we are going to BLAST our sequences from our PCR products and see if they
resemble any life forms that we know about on earth. If we do get a match this could
indicate that there is, or has been, life on Mars.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html
What do you need to do?
1. Open the Word file named “DNA Sequence Samples”.
2. Copy the DNA sequence “Sample 4”.
3. Maximise the internet browser and go the following website:http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
Choose the program called Nucleotide Blast, about half way down the page.
4. Paste the sequence into the box which says “Enter query sequence”
5. Scroll down the web page to where it says “Choose search set”. Here you can
choose what database to search in. You can choose from the Human Genome,
Mouse Genome or Others. Try the search with all three databases and see if any of
them give you a match.
6. Once you have chosen a search set, scroll down and click on the button that says
BLAST. Now the database is performing the search of your sequence against all of
the other sequences contained in the database.
7. Scroll down the page once you have your results.
What areas of Mars did you amplify DNA from?
Did you get a BLAST match?
What organism did your sequence match against?
What was the DNA sequence identified similar to?
Can you tell how similar the sequence you pasted in is to the sequence it matches?
Repeat this with the DNA sequence “Sample 2”.
What can you conclude about your 2 sequences?
If your DNA sequence is part of a gene that codes for a protein then we can identify
from the DNA sequence what the amino acid sequence is. Amino acids are the
building blocks of proteins.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html
Your first DNA sequence that you looked at has been translated into an amino acid
sequence and is written below. By looking at the amino acid sequence can you work
out which amino acids are present from the 1-Letter codes? There are quite a lot of
them!
ETSPSSIFTSASSAWASYSITSRMVMAILLVGSVLILVPPSARARRNMSLMTLSILCRSSRLE
TSISSSERS.RLELNASSVSPISVANGVLNSCARLALNWPSSGISTSFDRVTRTGQSRGEFLLQ
SPSLQAARQAAPETALRPGYSMHRSGPGP
Amino acid name
3-Letter code
1-Letter code
Valine
Val
V
TYrosine
Tyr
Y
Tryptophan
Trp
W
Threonine
Thr
T
Serine
Ser
S
Proline
Pro
P
Phenylalanine
Phe
F
Methionine
Met
M
Lysine
Lys
K
Leucine
Leu
L
Isoleucine
Ile
I
Histidine
His
H
Glycine
Gly
G
Alanine
Ala
A
Glutamic Acid
Glu
E
Glutamine
Gln
Q
Cysteine
Cys
C
Aspartic Acid
Asp
D
Asparagine
Asn
N
Arginine
Arg
R
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html