Chapter 21: Manipulating genomes

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21.1 DNA profiling and electrophoresis

Human genomes

A genome contains all of the alleles in an organism.

The Human Genome Project was an international research effort to determine the sequence of the human genome and identify the genes that it contains. The Project was coordinated by the National Institutes of Health and the U.S. Department of Energy. Additional contributors included universities across the United States and international partners in the United Kingdom, France, Germany, Japan, and China. The Human Genome Project formally began in 1990 and was completed in 2003.

Electrophoresis

Electrophoresis is basically genetic chromatography. It is a technique used to separate DNA fragments according to their size by measuring the length samples travel in the gel. Samples are loaded into wells (indentations) at the negative end of a gel (cathode), and an electric current is applied to pull them through the gel to the anode because the proteins are negatively charged. Longer proteins will travel slower as there is more resistance from the gel. Eco R1.

Uses of electrophoresis

Electrophoresis can be used to analyze proteins, including those found in human blood. Unlike DNA or RNA, these proteins may not be charged so the sample is usually treated with a detergent. Mixing blood with this detergent causes proteins to unravel and also imparts a negative charge to them. This makes the protein migrate towards the positive end (anode) in electrophoresis. The greater the charge, the further the protein will travel. This can be used to measure the presence of specific proteins in blood or the presence of abnormal substances or even the presence of diseases.

Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.

DNA profiling

This process was developed in Lesticer and was used to catch criminals. DNA samples can come from blood, skin, saliver, even one drop of blood is plenty to extract enough DNA. First done in the 1980s when many more samples were needed but today we have PCR (polymerase chain reaction) that is basically a DNA photocopier so as many copies of the DNA can be made as needed and technology is ore sophisticated. It has played an important role in the criminal justice system and other fields. important in forensics.

Extraction: hypervariable regions. For human DNA profiling, only selected sections of non-coding DNA are used. This is because...

Digestion: strands of DNA are cut into small fragments using restriction endonuclease enzymes such as eco R1. Different restriction endonucleases cut DNA at a specific nucleotide sequence known as a restriction site. Restriction endos give scientists the ability to cut the dna strands at defined points in the introns. a mixture of

Separation (gel electrophoresis): the DNA fragments are separated using gel electrop, the gel is then immersed in an alkali solution to separate the DNA double strands into single strands (the hydrogen bonds), which results in two single strands of DNA. This is done because a radio active pobe will be added so the probe will bond to a single strand of introns, if they are not singe stranded this will not work.

Transfer (southern blotting): This is when the DNA fragments are transferred from the gel onto a nylon membrane. a large slab of gel is more awkward to deal with than when the DNA is on a nylon membrane. A thin nylon membrane is laid over the gel and then covered with several sheets of absorbent paper which draw up the liquid bringing the DNA with it by capillary action. This transfs the dna frags to the nylon membrane in the exact same positions as in the gel.

Hybridisation: fluorescent (or radioactive back in the day) DNA probes are added in excess to the DNA fras on the membrane. The dna probes are short dna or rna sequenses complimentary to the dna sequences.

Development: the excess probes are washed of and the nylon membrane with the radioactively marked dna is placed onto an x-ray film or put under a UV light if it was marked that way. DNA probes identify the microsatellite regions

Further development: x-ray images taken off the membrane. the fragments show a pattern of bars (the DNA profile) which is unique for each individual (appart from identical twins).

Uses and ethical issues

The guy who developed it now has a knighthood. used also for paternity testing, evolutionary relationships to see how closely related different individulas and species are. Also genetic screening to test to see whether individuals have certsin genetic diseases, recesive genes may show up as a banding pattern. Police cannot have a bank of DNA from the public becasue it is a violation of privacy. This was done in sweden or finland but the DNA was sold to insurance companies who were interested to see things like how likely someone was to die from a heart attak.

Mitochondrial DNA

Mitochondrial DNA is passed from females (the Romanov family).

https://en.wikipedia.org/wiki/Mitochondrial_DNA

Polymerase chain reaction

PCR is like a DNA photocopier. It can be used for criminal investigation. also called a Thermal cycler. DNA is put into the machine as well as tack polymerase, primers and free nucleotides. The first stage is separating the strands

Separating the strands: temperature in the PCR machine is increased to around 90° for 30 seconds so the hydrogen bonds break and the DNA denatures.

Annealing of the primers: the temperature is decreased to 55-60° and the primers bind (anneal) to the ends of the DNA strands. They are needed for the replication of the strands to occur.

Synthesis of DNA: The temperature is increased again to 72-75° for at least a minute, this is the optimum temperature for Taq polymerase to work. DNA polymerase adds to the primer, building up complimentary strands of DNA and so producing double-stranded DNA identical o the original sequence. The enzyme Taq polymerase is used, which is obtained from thermophilic bacteria called Thermus aquaticus found in hot springs. This cycle is repeated so that many more copies of DNA can be made.

Uses of PCR (see harwood powerpoint)

Infection detection:

Paternity tests:

Detecting mutations:

Genetic screening:

Detection and diagnosis of cancer:

Tissue matching: in organ transplants

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21.2 DNA sequencing and analysis

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21.3 Using DNA sequencing

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21.4 Genetic engineering

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21.5 Gene technology and ethics

Gene therapy

Gene therapy is used to treat genetic disorders at the source byt altering the mutated alleles that cause them. There are two types of gene therapy: somatic, which alters the alleles in normal body cells, and germ line, which alters the alleles in the sex cells/zygote blastomere, though this technique is currently illegal.

Somatic

is used to tread the disease but does not prevemt it.

germ line

used to prevent the inheritance of the disease. this works by altering all fallowing cells.