, Human genome
The genome of an organism is the genetic material it contain- for
eukaryotes this is both DNA in the nucleus & mitochondria combined.
The large non-coding regions of DNA that are removed from mRNA
before it is translated into a polypeptide chain are known as introns. The
exons are the sections of DNA that code for proteins.
Within introns, telomeres & centromeres there are short sequences of
DNA that are repeated many times. This is known as satellite DNA.
In a region known as a minisatellite (variable number tandem repeats
VNTR), a sequence of 20-50 base pairs will be repeated 100’s of times.
A microsatellite (short tandem repeats (STR) is a smaller region of just
2-4 base pairs repeated 5-15 times.
These satellites always appear in the same positions on the
chromosomes, but the number of repeats of each micro- or minisatellite
varies between each individual, as different lengths of repeats are
inherited from both parents. Only monozygotic twins have an identical
satellite pattern.
, Producing a DNA profile
Producing an image of the patterns of DNA of an individual is known as DNA profiling.
1.Extracting the DNA- Using PCR
2.Digesting the sample- Restriction endonucleases cut DNA into fragments which include a
mixture of intact micro/mini satellite regions. Different restriction endonucleases cut DNA
at a specific nucleotide sequence- the restriction/recognition site. All restriction
endonucleases make 2 cuts, once through each strand of the DNA double helix.
3.Separating the DNA fragments- Separated using electrophoresis which is a type of
chromatography that relies on the way charged particles move through a gel under the
influence of an electric current. DNA is –vely charged & so moves from the cathode to the
anode. The gel slows the rate of movement of the fragments increasing separation. Smaller
fragments can move through the gel more easily than larger fragments so the smaller
fragments are found nearer the anode on a DNA profile. When the fastest smallest
fragments reach the anode end of the gel, the electric current is switched off & the gel is
immersed in an alkali. This denatures the DNA fragments which causes the 2 DNA strands of
each fragment to separate, exposing bases. The single stranded DNA fragments are then
transferred onto a nylon membrane by southern blotting.
4.Hybridisation- Radioactive or fluorescent DNA probes are added in excess to the DNA
fragments on the membrane. DNA probes are short DNA or RNA sequences complementary
to a known DNA sequence. Under particular conditions they bind to the complementary
strands of DNA. This is called hybridisation.
5.Seeing the evidence- If radioactive labels were added to the DNA probes, x-ray images
are taken of the membrane. If fluorescent labels were added to the DNA probes, the
membrane is placed under UV light so the fluorescent tags glow. Homozygous loci will have
identical alleles; with identical lengths; producing a single band on a DNA profile
, PCR- polymerase chain reaction
PCR allows a lot of DNA to be produced from the tiniest original sample. The DNA sample to
be amplified, an excess of the 4 nucleotide bases A, T, C & G, small primer DNA sequences &
the enzyme DNA polymerase are mixed in a vial
that is place in the PCR machine (thermal cycler).
Step 1 separating the strands The temp in
the PCR machine is ↑ to 90-95˚c for 30 secs.
This denatures the DNA by breaking the
hydrogen bonds holding the DNA strands
together so they separate.
Step 2 annealing of the primers The temp is
↓ to 55-60˚c & the primers anneal to the ends of
the DNA strands. Primers allow DNA polymerase
to bind for replication
Step 3 synthesis of DNA The temp is ↑ to
72-75˚c for at least 1 min. this is the optimum
temp for DNA polymerase to work best. DNA
polymerase adds bases to the primer, building up
complementary strands of DNA & so producing
double stranded DNA identical to the original
sequence. The enzyme Taq
polymerase is used, which is obtained
from thermophilic bacteria found in
hot springs.
Step 4 the cycle is repeated
The genome of an organism is the genetic material it contain- for
eukaryotes this is both DNA in the nucleus & mitochondria combined.
The large non-coding regions of DNA that are removed from mRNA
before it is translated into a polypeptide chain are known as introns. The
exons are the sections of DNA that code for proteins.
Within introns, telomeres & centromeres there are short sequences of
DNA that are repeated many times. This is known as satellite DNA.
In a region known as a minisatellite (variable number tandem repeats
VNTR), a sequence of 20-50 base pairs will be repeated 100’s of times.
A microsatellite (short tandem repeats (STR) is a smaller region of just
2-4 base pairs repeated 5-15 times.
These satellites always appear in the same positions on the
chromosomes, but the number of repeats of each micro- or minisatellite
varies between each individual, as different lengths of repeats are
inherited from both parents. Only monozygotic twins have an identical
satellite pattern.
, Producing a DNA profile
Producing an image of the patterns of DNA of an individual is known as DNA profiling.
1.Extracting the DNA- Using PCR
2.Digesting the sample- Restriction endonucleases cut DNA into fragments which include a
mixture of intact micro/mini satellite regions. Different restriction endonucleases cut DNA
at a specific nucleotide sequence- the restriction/recognition site. All restriction
endonucleases make 2 cuts, once through each strand of the DNA double helix.
3.Separating the DNA fragments- Separated using electrophoresis which is a type of
chromatography that relies on the way charged particles move through a gel under the
influence of an electric current. DNA is –vely charged & so moves from the cathode to the
anode. The gel slows the rate of movement of the fragments increasing separation. Smaller
fragments can move through the gel more easily than larger fragments so the smaller
fragments are found nearer the anode on a DNA profile. When the fastest smallest
fragments reach the anode end of the gel, the electric current is switched off & the gel is
immersed in an alkali. This denatures the DNA fragments which causes the 2 DNA strands of
each fragment to separate, exposing bases. The single stranded DNA fragments are then
transferred onto a nylon membrane by southern blotting.
4.Hybridisation- Radioactive or fluorescent DNA probes are added in excess to the DNA
fragments on the membrane. DNA probes are short DNA or RNA sequences complementary
to a known DNA sequence. Under particular conditions they bind to the complementary
strands of DNA. This is called hybridisation.
5.Seeing the evidence- If radioactive labels were added to the DNA probes, x-ray images
are taken of the membrane. If fluorescent labels were added to the DNA probes, the
membrane is placed under UV light so the fluorescent tags glow. Homozygous loci will have
identical alleles; with identical lengths; producing a single band on a DNA profile
, PCR- polymerase chain reaction
PCR allows a lot of DNA to be produced from the tiniest original sample. The DNA sample to
be amplified, an excess of the 4 nucleotide bases A, T, C & G, small primer DNA sequences &
the enzyme DNA polymerase are mixed in a vial
that is place in the PCR machine (thermal cycler).
Step 1 separating the strands The temp in
the PCR machine is ↑ to 90-95˚c for 30 secs.
This denatures the DNA by breaking the
hydrogen bonds holding the DNA strands
together so they separate.
Step 2 annealing of the primers The temp is
↓ to 55-60˚c & the primers anneal to the ends of
the DNA strands. Primers allow DNA polymerase
to bind for replication
Step 3 synthesis of DNA The temp is ↑ to
72-75˚c for at least 1 min. this is the optimum
temp for DNA polymerase to work best. DNA
polymerase adds bases to the primer, building up
complementary strands of DNA & so producing
double stranded DNA identical to the original
sequence. The enzyme Taq
polymerase is used, which is obtained
from thermophilic bacteria found in
hot springs.
Step 4 the cycle is repeated
