Download Restriction Fragment Length Polymorphisms

DNA technology uses:
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Genetic engineering
Genetically modified (GM) crops
Gene Therapy
Genetic (DNA) Fingerprinting
Genetic Screening
Personalised medicine
Techniques used in Dna tech
• Amplifying DNA: PCR
• Comparing DNA: identifying and using
specific marker sites using restriction
endonuclease enzymes & DNA probes
PCR is used in:
• forensic science e.g. genetic fingerprinting;
• medical research e.g. genetic screening to find
alleles for genetic diseases;
• scientific research e.g. to investigate DNA of extinct
species to determine their relationship with living
organisms;
• The Human Genome Project.
Its purpose is to amplify very small quantities of DNA
producing, many identical copies of the selected
section of DNA in a very short time.
method
• Heat to 95oC to break hydrogen bonds between
complementary bases and separate strands
• Cool to 40-60oC & add excess primer
Primers are short single stranded sequences of about 20
nucleotides which are complementary to bases in part of the
DNA strand being copied.
Cooling allows the primers to bind (anneal) to each DNA
strand at specific complementary points
– Primers stop the DNA strands rejoining
– They bracket the DNA to be copied by attaching to the
end of the DNA sequence and shows DNA polymerase
where to start copying. There are 2 different primers as
there are 2 different ends of the DNA
– DNA replication can only start with a double stranded
region
• Heat to 70oC. Add free DNA nucleotides &
thermostable DNA polymerase
Polymerase copies each strand , starting with the
primers.
The polymerase is thermostable allowing the
process to occur rapidly at the higher temperatures
required, without denaturing. The enzyme was
originally isolated from a bacterium that lives in hot
springs.
• TWO DNA molecules will be formed following PCR
and are used as templates as the process is
repeated. This allows millions of copies of the
original DNA to be produced in a very short time.
HEAT 70
95 C
o
o
primer
Cool 40-60 C
o
DNA polymerase
primer
AVOID CONTAMINATION
with any other DNA source
as both the original & contaminated DNA will
be amplified
The Polymerase Chain Reaction sumanisinc
brill animation
http://www.maxanim.com/genetics/PCR/pcr.swf
www.dnalc.org/shockwave/pcranwhole.html/south
an.html
sections of DNA
that are used to identify
specific DNA sequences
Allows for comparison of DNA
sequences between different people
•Enzymes that recognise & cut specific
nucleotide sequences.
•Originally from bacteria that use RE
enzymes to cut DNA of attacking
bacteriophages.
e.g.
EcoR1
recognises the sequence GAATTC, cutting
between the G & A of each strand
produces sticky ends because G & A don’t
lie opposite each other.
3’ CCCTAGGAATTCAGACC
CCCTAGG AATTCAGACC 5’
GTCTGG 3’
3’
5’ GGGATCCTTAA
GGGATCCTTAAGTCTGG
sticky ends
e.g.
Bam1
recognises the sequence GGATCC, cutting
between the G & G of each strand
produces sticky ends because G & G don’t
lie opposite each other.
…G G A T C C…
…C C T A G G…
e.g.
Hae 111
recognises the sequence GGCC, cutting between
the G & C of each strand
produces blunt ends because G & C lie opposite
each other, these are not as useful as sticky ends.
…G G C C…
…C C G G…
•Restriction enzymes will cut DNA into a
number of fragments, depending upon the
number of recognition sites in the sample of
DNA.
•The size of the fragment depends on how
far apart the recognition sites are.
•If DNA has 4 recognition sites, 5 fragment
lengths of DNA are produced.
5 fragments
Recognition
site 4
Recognition
site 3
Recognition
site 2
Recognition
site 1
DNA SAMPLE
•If two samples of DNA are identical,
treatment with a particular restriction
enzyme will produce the same number of
DNA fragments, each of similar size.
•However, different individuals have
different DNA sequences and therefore
produce different fragments when a
particular restriction enzyme is used to cut
an equivalent section of DNA.
•The fragments produced are called RFLPs,
RESTRICTION FRAGMENT LENGTH
POLYMORPHISMS.
PERSON A
Recognition
site 4
Recognition
site 4
Recognition
site 5
Recognition
site 1
Recognition
site 2
Recognition
site 3
Recognition
site 3
Recognition
site 2
Recognition
site 1
DNA SAMPLE
5 fragments
PERSON B
DNA SAMPLE
6 fragments
This allows for comparison or identification of DNA samples
pronounced RiFlips
Restriction Fragment Length
Polymorphisms
http://highered.mcgraw-hill.com/olc/dl/120078/bio20.swf
SNPs
MRSs
pronounced snips
Single Nucleotide Polymorphisms
A SNP is ……
a difference in a single nucleotide in a section of
DNA
It can give rise to different alleles for a gene
egs include sickle cell anaemia (chromosome 11)
Sickle cell anaemia
normal Hb chain
Amino acid
Thr
sequence
DNA base
...ACT
sequence
codon number
4
sickle cell Hb chain
Amino acid
Thr
sequence
DNA base
...ACT
sequence
Pro
Glu
Glu
CCT
GAG
GAG...
5
6
Pro
Val
CCT
GTG
7
Glu
GAG...
Chromosome 16
Genetically
Ginger
RSci Advent
gcsbio.weebly.
com
Microsatellite Repeat Sequences
(Also called variable number tandem repeats – VNTRs).
• Only approximately 2% DNA codes
for proteins and 3% has a
regulatory role. The remaining 95%
has unknown function and is called
non-coding DNA.
• Within this DNA there are many
sections where a small number of
bases are repeated many times
e.g. ….ATCATCATCATCATCATCATC…
• These sequences are called
microsatellite repeat sequences or
short tandem repeats.
• The number of repeats of these
MRSs are unique to an individual.
Short sequences of single stranded DNA
with known nucleotide and base sequence.
May be fluorescent or radioactively
labelled, to enable it to be located.
Radioactive probes contain radioactive
isotopes of nitrogen in their nucleotide
structure.
•The DNA is treated to separate the
DNA strands (heat)
•Add probe – it base pairs with
complementary DNA sequence.
•Detect the target sequence:
• Fluorescent probe use a laser scanner
• Radioactive probe use X-ray film
•They are used to identify specific gene
sequences .
•Many inherited diseases are caused by a
single allele with a known base sequences
•A probe complementary to the allele
nucleotide sequence is used:
• If it binds the disease is present
• If it doesn’t bind it is not present.
PGD
Pre implantation genetic diagnosis
This is the use of DNA probes to detect
defective genes in an embryo to reduce the
chance of a hereditary disease in a family.
Measuring genetic variation
allows the following questions to
be answered:
1. Does a particular gene differ from person to
person? Are two persons closely related?
2. Are certain alleles associated with an hereditary
disease?
3. How do genes vary within a population? How
much genetic diversity is there within a
particular species?
4. How do genes differ from species to species?
How can this be used to unravel the taxonomic
relationship among species?
Genetic (dna) fingerprinting
1. Extract DNA from a sample
e.g. from a crime scene, often PCR required to
amplify the DNA
2. Restriction Enzymes cut the DNA into different
sized fragments
Specific MRSs used as they can be measured
more accurately than RFLPs. Primers used to
flank the MRS region involved to ensure
fragments only come from that region
3. DNA fragments separated using gel
electrophoresis.
DNA is placed into separate wells in agar gel.
An electric current is applied across the length
of the gel, with the negative electrode closest
to the wells of DNA.
DNA has a negative charge, so moves through
the spaces in the gel towards the positive
electrode.
Smaller DNA fragments move further than
larger fragments.
4. DNA is heated to separate the DNA strands to
make it single stranded.
5. The gel will dry up, so the DNA fragments are
transferred to a nylon membrane (process
called Southern blotting)
Radioactive or fluorescent DNA probes are
added to the membrane and will join to the
complementary base sequences in the
microsatellite region selected. The sample is
washed to remove any probes that do not base
pair.
5. The labelled DNA is added to X-ray film or a
laser scanner is used to detect the radioactive
or fluorescent probes and a pattern of bars
appear, revealing the position of the DNA
fragments: a DNA fingerprint.
Diagram p82