manipulating genomes Flashcards
1
Q
What is the genome of an
organism?
A
All of the genetical material • For eukaryotes, it is the DNA in the nucleus and the mitochondria • Only 2% of your total DNA codes for proteins (exons) • The large non-coding regions of DNA that are removed from mRNA before it is translated are called introns
2
Q
What is satellite DNA?
A
Short sequences of DNA that are
repeated many times within introns,
telomeres and centromeres
3
Q
What is a minisatellite?
A
A sequence of 20-50 base pairs that will be repeated from 50 to several hundred times • Occur at more than 1000 location in the human genome • Also known as variable number tandem repeats (VNTRs
4
Q
What is a microsatellite?
A
A region of 2-4 bases repeated only
5-15 times
• Also known as short tandem
repeats (STRs)
5
Q
What are the similarities and
differences in satellites in
different people?
A
Always appear in the same positions on the chromosomes • The number of repeats of each mini or microsatellite varies between individuals, as different lengths of repeats are inherited from both parents
6
Q
What is DNA profiling?
A
Producing an image of the patterns in the DNA of an individual • A technique employed by scientists to assist in the identification of individuals or familial relationships
7
Q
What is gel electrophoresis?
A
A technique used to separate cut fragments of DNA • Uses the way charged particles move through a gel medium under the influence of an electric current • The gel is then immersed in alkali in order to separate the DNA double strands into single strands • The single-stranded DNA fragments are then transferred onto a membrane by Southern blotting
8
Q
What are the stages in
producing a DNA profile?
A
1. Extracting the DNA DNA is extracted from a tissue sample. Using the polymerase chain reaction (PCR), the tiniest fragment of tissue can give scientists enough DNA to develop a profile 2. Digesting the sample The strands of DNA are cut into small fragments using restriction endonuclease enzymes. They are cut at a specific nucleotide sequence called a restriction/ recognition site. 2 cuts are made, one through each strand of the DNA double helix. 3. Separating the DNA fragments The cut fragments of DNA are separated to form a clear, pattern, using gel electrophoresis 4. Hybridisation Radioactive or fluorescent DNA probes are added in excess to the DNA fragments on the membrane. Theses are short DNA or RNA sequences complementary to a known DNA sequence. They bind to the complementary strands of DNA and identify the microsatellite regions 5. Seeing the evidence X-ray images can be taken if radioactive labels where added to the DNA probes, and UV can be used if fluorescent labels were added
9
Q
What is the Polymerase chain
reaction (PCR)?
A
A version of the natural process by
which DNA is replicated, and allows
scientists to produce a lot of DNA
from the tiniest original sample
10
Q
How is the PCR machine used?
A
(aka a thermal cycler) • Temperature is carefully controlled and changes rapidly at programmed intervals • The reaction can be repeated many times by the PCR machine, which cycles through the programmed settings • 30 repeats gives around 1 billion copies of the original DNA sample, which is more than enough to carry out DNA profiling
11
Q
What are the steps involved in
the PCR?
A
1. Separating the strands • Temperature in the PCR machine is increased to 90-95°C for 30 seconds • This denatures DNA by breaking the hydrogen bonds holding the DNA strands together so they separate 2. Annealing of the primers • Temperature is decreased to 55-60°C and the primers bind (anneal) to the ends of the DNA strands • They are needed for the replication of the strands to occur 3. Synthesis of DNA • Temperature is increased to 72-75°C for at least 1 minute, as this is the optimum temperature for DNA polymerase • DNA polymerase adds bases to the primer, building up complementary strands of DNA and 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
12
Q
What are the uses of DNA
profiling?
A
• Forensic science and criminal investigations • Proving the paternity of a child or in immigration cases to prove or disprove family relationships • To identify the species to which an organism belongs and to demonstrate evolutionary relationships between different species • To identify individuals who are at risk of developing particular diseases
13
Q
Give a brief history of DNA
Sequencing
A
1970s • Sanger sequencing enabled Fredrick Sanger and his team to read sequences of 500-800 bases at a time • Technique involved radioactive labelling of bases and gel electrophoresis on a single gel 1990 • Human Genome Project was established (HGP) • Scientist from a number of countries worked to map the entire human genome, making the data freely available to scientist all over the world • The first complete human genome sequence was published in 2003
14
Q
How does DNA sequencing
work?
A
• DNA is chopped into fragments, and each fragment is sequenced • The process involves terminator bases which are modified versions of A, C, T and G, which stop DNA synthesis when they are included • An A terminator will stop DNA synthesis at the location that an A base would be added etc • A is green, G is yellow, T is red, C is blue
15
Q
Describe the sequencing
process (capillary method)
A
1. Same process as in DNA profiling to produce double-stranded DNA. Mix DNA with a primer, DNA polymerase, an excess of normal nucleotides, and terminator bases 2. When a terminator base is added instead of a normal nucleotide, the synthesis of DNA is terminated. This results in many DNA fragments of random different lengths. After many cycles, all of the possible DNA chains will be produced with reactions stopped at every base. The fragments are separated according to their length by capillary sequencing. Lasers detect the different colours of the fluorescent markers on the terminator bases and thus the order of the sequence. 3. The order of bases in the capillary tubes shows the sequence of the new, complementary strand of DNA, which is used to build up the sequence of the original DNA strand. The data is them fed into a computer that reassembles the genomes by comparing all the fragments and finding the areas of overlap between them
16
Q
Computer analysis of all data
to give original DNA sequence
A
17
Q
What is capillary sequencing?
A
• Works like gel electrophoresis in
minute capillary tubes
18
Q
Describe next-generation
sequencing
A
• Instead of using a gel or capillaries, the sequencing reaction takes place on a plastic slide called a flow cell • Millions of DNA fragments are attached to the slide and replicated in situ using PCR to form clusters of identical DNA fragments • Sequencing process still uses a principle of adding a coloured terminator base to stop the reaction so an image can be taken • Known as ‘massively parallel sequencing’ as all of the clusters are being sequenced and imaged at the same time
19
Q
What is bioinformatics?
A
The development of the software and computing tools needed to organise and analyse raw biological data • Includes the development of algorithms, mathematical models, and statistical tests
20
Q
What is computational
biology?
A
The use of data from bioinformatics to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances • The study of biology using computational techniques, especially in the analysis of huge amounts of biodata • Helps us use the information from DNA sequencing e.g. in identifying genes linked to specific diseases
21
Q
What are genome-wide
comparisons used for?
A
Analysing the human genome • Analysing the genomes of pathogens • Identifying species (DNA barcoding) • Searching for evolutionary relationships
22
Q
How is the human genome
analysed?
A
• Since the first complete draft of the human genome was published in 2003, research projects e.g. the 100,000 Genome Project, have sequenced human genomes • Computers can analyse and compare the genomes of many individuals, revealing patterns in the DNA we inherit and the diseases to which we are vulnerable
23
Q
What does the analysis of the
genomes of pathogens allow?
A
Doctors to find out the source of an infections. e.g. bird flu or MRSA in hospitals • Doctors to identify antibioticresistant strains of bacteria, ensuring antibiotics are only used when they will be effective • Scientists to track the progress of an outbreak of a potentially serious disease and monitor potential epidemics, e.g. flue and Ebola • Scientists to identify regions in the genome of pathogens that may be useful targets in the development of new drugs, and to identify genetic markers for use in vaccines
24
Q
How is analysis used in
identifying species (DNA
barcoding)?
A
• Using traditional methods of observation, it can be very difficult to determine which species an organism belongs to, or if a new species has been discovered • In the International Barcode of Life (iBOL) project, scientists identify species using relatively short sections of DNA from a conserved region of the genome
25
Q
Which region of DNA is used to
analyse the animal species,
and why?
A
• A 648 base-pair sectional the mitochondrial DNA in the gene cytochrome oxidase, that codes for an enzyme involved in cellular respiration • The section is small enough to be sequenced quickly and cheaply • Varies enough to give clear differences between species
26
Q
Why isn’t the same region of
DNA used to analyse land
plants?
A
That region of DNA doesn’t evolve
quickly enough in land plants to
show clear differences between
species
27
Q
What region of DNA is used to
analyse land plant species?
A
Two regions in the DNA of
chloroplasts are used
38
Q
What are the benefits of using
restriction endonucleases?
A
• Many restriction endonucleases cut the 2 DNA strands unevenly, leaving 1 of the strands a few bases longer than the other strand • These region with unpaired, exposed bases are called ‘sticky ends’ • These sticky ends make it easier to insert the desired gene into the DNA of a different organism
43
Q
Why are plasmids that are used as
vectors usually given a second
marker gene?
A
It is used to show that the plasmid contains the recombinant gene • The marker gene is placed in the plasmid by genetic engineering • The plasmid is then cut by a restriction enzyme within this marker gene to insert the desired gene • If the DNA fragment is intent successfully, the marker gene will not function • e.g. marker genes may be for producing fluorescence, or an enzyme that causes a colour change in the presence of a particular medium, instead of for antibiotic resistance (which was used in the early days)
