6.3 MANIPULATING GENOMES Flashcards
What is the human genome project?
The human genome project:
- started in 1990, finished in 2003
- technology now existed to identify people using samples of DNA and to sequence sections of DNA
- however scientists wanted to know the entire sequence so the HGP was born
- estimated to take 15 years, took 13 years
- estimated cost US$3 billion
What were the aims of the human genome project?
Aims of HGP:
- to identify all the approximately 20,000-25,000 genes in human DNA
- to find where each gene is located
- to determine the sequences of the 3 billion chemical base pairs that make up human DNA
- to store this information in databases
What is a genome?
A genome is all the genetic material of an organism.
What are introns?
Introns are regions of non-coding DNA or RNA.
What are exons?
`Exons are regions of DNA that code for proteins.
What is VNTR?
VNTR stands for variable number tandem repeats, a minisatellite is a sequence of 20-50 base pairs that is repeated 50 to several 100 times.
What is STR?
STR stands for short tandem repeats, a microsatellite is a small region of 2-4 bases repeated 5-15 times.
What is DNA profiling?
DNA profiling produces an image of the patterns in the non-coding DNA of an individual.
What is polymerase chain reaction?
PCR is a process by which a small sample of DNA can be amplified using specific enzymes and temperature changes.
What are restriction endonucleases?
Restriction endonucleases are enzymes that chop a DNA strand into lots of small pieces.
What is electrophoresis?
Electrophoresis is a type of chromatography that relies on the way charged particles move through a gel under the influence of an electrical current. used to separate nucleic acid fragments.
What is hybridisation?
Hybridisation is the addition of fluorescent or radioactive probes to DNA fragments via complementary base pairing.
What is a telomere?
A telomere is the structure at the end of a chromosome.
What is a histone?
Histones are proteins that form a complex with DNA called chromatin.
What are the applications of PCR?
Applications of PCR:
- detection of oncogenes = detect type of mutation leading to cancer
- detecting mutations = detecting genetic diseases
- identify viral infections = verify the type of viral infection present
- monitoring the spread of infectious diseases = also monitoring the emergence of new strains
- forensic science = small quantities of DNA can be amplified to identify chemicals or ascertain percentage
- research = amplifying DNA from extinct organisms e,g mammoths
- tissue typing = donor and recipient tissues can be matched to reduce the risk of infection
What is PCR?
PCR:
- cyclic reaction
- a method of copying DNA fragments
- automated method (rapid and efficient)
- requires small DNA sample, DNA polymerase (taq (thermos aquatecus) DNA polymerase-found in thermophilic bacteria-heat resistant, more resistant to denaturing), primers, nucleotides, thermocycler
What are the steps of PCR?
Steps of PCR:
1. separation of DNA strands (denaturation)
- DNA fragments, primers, DNA polymerase and nucleotides added to a thermocycler
- 95 degree C temp causes DNA strands to separate (denature)
2. addition of primers (annealing)
- primers anneal (join) to their complimentary bases (55-68 degrees C)
3. synthesis of DNA (new DNA strands formed)
- temp increased to 72 degrees C, the optimum temp of DNA polymerase
- DNA polymerase attaches nucleotides to along each of the separated DNA strands
What are the advantages of PCR?
Advantages of PCR:
very rapid- billions of copies can be made in hours with a minute amount of DNA (e.g crime scene)
does not require living cells- only requires a bases sequence, no complex culturing required
What are the uses of DNA probes?
Uses of DNA probes:
- locate a specific gene for genetic engineering
- identify the same gene in a variety of different genomes from different species when conducting genome comparisons
- identify the presence or absence of a specific allele for a particular genetic disease or one that gives susceptibility to a particular condition
What does gel electrophoresis do?
Electrophoresis:
- separates different lengths of DNA
- phosphates in the backbone of DNA are negatively charged
- DNA fragments are placed in walls at the top of an agar gel
- an electric current in applied over it
- agar is usually a ‘mesh’ which resist the movement of DNA fragments through it
- the DNA moves towards the positive electrode, but at different rates
- small fragments get there quicker (travel furthest)
What is the process of gel electrophoresis?
Gel electrophoresis process:
1. extraction of DNA from sample
2. fragmentation of DNA using restriction endonuclease enzyme
3. separation using electrophoresis (small fragments travel furthest)
4. these bands are colourless but are revealed using a radioactive or fluorescent probe
What do DNA ladders tell us about samples produced by PCR?
DNA ladders show known lengths of DNA fragments.
e.g DNA making up light band size is about 500 bp (base pairs)
What is DNA profiling?
DNA profiling from satellite DNA:
- introns, centromeres and telomeres have short sequences of DNA that are repeated many times called a variable number tandem repeat (VNTR)
- these appear at defined locations on each chromosome but differ in length for each individual, so everyone will have their own satellite pattern (unless they are identical twins)
- producing an image of these patterns = DNA profiling
What is the procedure of DNA profiling?
DNA profiling procedure (creating a DNA profile):
1. extract DNA from sample- can use PCR if the sample is small
2. digest the sample- DNA cut into small fragments with restriction endonuclease. variety of these used to cut at defined points within intron that leaves satellite intact (VNTR)
3. separate DNA fragments using electrophoresis
4. DNA is separated into singe strands using an alkaline buffer solution
5. DNA fragments transferred to nylon membrane by southern blotting
6. hybridisation DNA probes added to label the fragments (radioactive/fluorescent). DNA probes identify the satellites are they have complimentary sequences
7. development- placed onto x-ray film and developed (dark bands where DNA probs are) or membrane placed under UV light if fluorescent tags have been used
What are the uses of DNA profiling?
Uses of DNA profiling:
- maternity and paternity disputes
- forensic science = convictions of crimes, proving innocence, identify victims body parts, identify descendants of soldiers killed in WWII, identify Nazi war criminals in South Africa, used in immigration cases to prove or disprove family relationships
During electrophoresis, why is the agarose gel placed into a buffering solution?
The agarose gel is placed into a buffering solution to maintain constant pH.
What is the purpose of a DNA ladder?
DNA ladders show known base pair fragment distances so unknown fragments can be compared to the DNA ladder and identified
Why do DNA molecules move towards the positive anode in an electrophoresis tank?
DNA molecules move towards the anode in an electrophoresis tank as DNA molecules are negatively charged (due to the negative charge on the phosphate group) so they are attracted to the oppositely charged cathode
What does the rate of movement depend on within an agarose gel?
Within agarose gel, the size of the DNA fragment will determine how far it travels. Due to the ‘mesh like’ property of the agar gel, small fragments travel further.
When the gel has finished running why is it placed in an alkaline buffer? (Electrophoresis)
When the gel has finished running it is placed into an alkaline buffer to denature the alkaline DNA and expose its bases.
What is southern blotting? (Electrophoresis)
Southern blotting is when strands are transferred to nitrocellulose paper/nylon membrane. The membrane is covered with several sheets of dry absorbent paper which draws alkaline solution (containing DNA) through the membrane.
How are genetic disorders diagnosed?
To identify certain genes that may cause genetic disorders, we can use DNA probes and DNA hybridisation.
What are uses of DNA probes?
Uses of DNA probes:
DNA probes are small fragments of nucleic acids (either cloned or artificially synthesised) that is labelled with an enzyme, a radioactive tag or a fluorescent tag
fluorescent dye tag - shows up as fluorescent bands when gel is exposed to a UV light source
radioactive tag - shows up as a dark band when the gel is exposed to photographic film
- the probe will bind to a complimentary DNA sequence by base pairing, identifying the presence and location of the target DNA sequence for further analysis
- a DNA probe is 50-80 nucleotides long made with complimentary bases to the gene of interest
- DNA strands are separated
- separated strands are mixed with DNA probe
- DNA probe binds to complimentary bases
- location can be identified by radioactivity/fluorescence of DNA sample
- need to know the base sequence of the gene first or presence can be identified
What is genetic screening?
Genetic screening:
- many genetic disorders are the result of a mutation
- if the mutation occurs in a dominant allele, all individuals who possess this allele will have the disorder
- if the mutation occurs in a recessive allele, heterozygous individuals will be carriers of the disease, they could pass the disease to their offspring if their partner is also heterozygous or homozygous recessive
- genetic councillors advise parents of the likelihood of their children having the disease . uses family history of a genetic disease
What is Sanger sequencing?
Sanger sequencing:
- four different terminators used (A, C, T and G). each terminator is a modified nucleotide that cannot polymerise to a subsequent nucleotide
- four reactions are run, in each reaction you have the DNA being sequenced, a mixture of ‘normal nucleotides’, one type of terminator nucleotide, a primer, DNA polymerase
- each tube contains millions of copies of the DNA template, nucleotides, and a good supply of the specific terminator nucleotide
- due to this, you get a variety of ‘partially completed’ DNA strands, because they have been ‘terminated’ at different points
How is Sanger sequencing done now?
Sanger sequencing now:
- terminator nucleotides are labelled with fluorescent dyes (instead of radioactive isotopes)
- the fragments are run through a single long thin tube containing a gel matrix in a process called capillary gel electrophoresis
- short fragments move quickly and long fragments move more slowly
- at the end of tube, its illuminated by a laser, allowing the attached dye to be detected
What are the uses and limitations of Sanger sequencing?
Uses and limitations of Sanger sequencing:
- typically used to separate pieces of DNA such as plasmids or DNA copied in PCR
- however, this method is expensive and inefficient for larger scale projects
- new large scale sequencing techniques are faster and less expensive = next generation sequencing
- the human genome can now be sequenced in a day
