Manipulating Genome Revision Notes
DNA Profiling
Human Genome
-genome is all the genetic material of an organism (DNA and mitochondria combined)
-chromosomes are made up of hundreds of millions of base pairs
-genes (regions that code for proteins) make up 2% of the genome
-the 98% are exons (non-coding region of DNA) (removed from mRNA before it is translated into a mature mRNA polypeptide chain)
-role of exons are unknown
Satellite DNA
-within introns, telomeres and centromeres there are short sequences of DNA that are repeated many times
-satellites appear in the same position on the chromosomes
-number of repeats of both mini-satellite and micro-satellite vary between individuals as different lengths of repeats are inherited from
individuals
-identical twins will have identical satellites area and closely related family members with have similar satellite areas
Mini-satellite Region Micro-satellite Region
-sequence of 20-50 base pairs -sequence of 2-4 base pairs
-repeated from 50 to several hundred times -repeated only 5-15 times
-occur at 1000 locations in the human genome -known as short tandem repeats (STR)
-can be known as variable number tandem repeats (VNTR)
Producing a DNA Profile
1) Extracting the DNA
-DNA must be extracted from tissue sample
-Tiniest amount of DNA is needed as it can be amplified by a technique called the polymerase chain reaction
2) Digesting the DNA
-DNA cut into different sized fragments using specialised enzymes called restriction endonucleases
-Different restriction endonucleases cut DNA at a specific nucleotide sequences known as the recognition site
-All restriction endonucleases makes 2 cuts – one through each DNA strand leaving either sticky or blunt ends
-Restriction endonucleases are used to cut DNA strands at defined points in the introns so the satellites are left
intact
3) Separating the DNA
-DNA fragments can be separated by a technique called electrophoresis
-negatively charged DNA moves through a gel medium towards the anode (positive electrode)
-smaller fragments move quicker than larger ones
-gel is immersed in alkali to separate the DNA double stands into single strands
-single stranded DNA fragments are transferred onto a membrane by Southern blotting
4) Hybridisation
-radioactive or fluorescent DNA probes are added in excess to the DNA fragments on the membrane
-probes are DNA/RNA sequences that are complementary to a known DNA sequence
-they bind to the complementary strands under certain temperatures and pH
-DNA probes help to identify the satellite regions
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 probe, the membrane is placed under UV light and the labels glow
-the fragments give a pattern of bars which is unique to each individual – DNA profile
Applications of DNA Profiling
Forensic Science – identify criminals when DNA is left at a crime scene, identify body parts of victims in terrorist attacks
Maternity and Paternity Disputes – half the short tandem repeats come from each parent so DNA profiles can be compared
Analysis of Disease – varying number of repeat sequences can be shown by electrophoresis (detect Huntington’s disease)
DNA Profiling
Human Genome
-genome is all the genetic material of an organism (DNA and mitochondria combined)
-chromosomes are made up of hundreds of millions of base pairs
-genes (regions that code for proteins) make up 2% of the genome
-the 98% are exons (non-coding region of DNA) (removed from mRNA before it is translated into a mature mRNA polypeptide chain)
-role of exons are unknown
Satellite DNA
-within introns, telomeres and centromeres there are short sequences of DNA that are repeated many times
-satellites appear in the same position on the chromosomes
-number of repeats of both mini-satellite and micro-satellite vary between individuals as different lengths of repeats are inherited from
individuals
-identical twins will have identical satellites area and closely related family members with have similar satellite areas
Mini-satellite Region Micro-satellite Region
-sequence of 20-50 base pairs -sequence of 2-4 base pairs
-repeated from 50 to several hundred times -repeated only 5-15 times
-occur at 1000 locations in the human genome -known as short tandem repeats (STR)
-can be known as variable number tandem repeats (VNTR)
Producing a DNA Profile
1) Extracting the DNA
-DNA must be extracted from tissue sample
-Tiniest amount of DNA is needed as it can be amplified by a technique called the polymerase chain reaction
2) Digesting the DNA
-DNA cut into different sized fragments using specialised enzymes called restriction endonucleases
-Different restriction endonucleases cut DNA at a specific nucleotide sequences known as the recognition site
-All restriction endonucleases makes 2 cuts – one through each DNA strand leaving either sticky or blunt ends
-Restriction endonucleases are used to cut DNA strands at defined points in the introns so the satellites are left
intact
3) Separating the DNA
-DNA fragments can be separated by a technique called electrophoresis
-negatively charged DNA moves through a gel medium towards the anode (positive electrode)
-smaller fragments move quicker than larger ones
-gel is immersed in alkali to separate the DNA double stands into single strands
-single stranded DNA fragments are transferred onto a membrane by Southern blotting
4) Hybridisation
-radioactive or fluorescent DNA probes are added in excess to the DNA fragments on the membrane
-probes are DNA/RNA sequences that are complementary to a known DNA sequence
-they bind to the complementary strands under certain temperatures and pH
-DNA probes help to identify the satellite regions
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 probe, the membrane is placed under UV light and the labels glow
-the fragments give a pattern of bars which is unique to each individual – DNA profile
Applications of DNA Profiling
Forensic Science – identify criminals when DNA is left at a crime scene, identify body parts of victims in terrorist attacks
Maternity and Paternity Disputes – half the short tandem repeats come from each parent so DNA profiles can be compared
Analysis of Disease – varying number of repeat sequences can be shown by electrophoresis (detect Huntington’s disease)
