6.21 - Manipulating genomes Flashcards
(41 cards)
1
Q
genome
A
all the genetic material an organism contains. Includes the base sequence in the nucleus and in mitochondria
2
Q
exons
A
coding regions of DNA
3
Q
introns
A
non-coding regions of DNA. Removed from mRNA before it is translated into a polypeptide chain. Contains satellite DNA
4
Q
DNA profiling
A
producing an image of the patterns of DNA in an individual to help identify an individual in forensics or determine familial relationships through the similarity of satellite DNA patterns
5
Q
Satellite DNA patterns
A
- short sequences of DNA that are repeated many times
- always appear in the same positions on each chromosome
- the number of repeats of each minisatellite or microsatellite DNA varies between each person
- only identical twins have the same satellite pattern
6
Q
VNTRs (minisatellites)
A
variable number tandem repeats
20-50 base pairs repeated 50 to several hundred times
7
Q
STRs (microsatellites)
A
short tandem repeats
2-4 base pairs repeated 5-15 times
8
Q
What is gel electrophoresis
A
- a technique used to separate DNA fragments according to their size
- DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel.
- DNA fragments are negatively charged, so they move towards the positive electrode
- Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones.
- When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each representing a group of same-sized DNA fragments.
9
Q
how to produce a DNA profile
A
- Extraction
DNA is extracted from the sample. DNA from a small sample can be replicated using PCR - Digestion
The strands of DNA are cut into small fragments by restriction endonucleases. Scientists use a mixture of endonucleases that cut DNA at specific points that leave the satellite DNA intact - Separation
The fragments of DNA are separated to form a clear recognisable pattern using gel electrophoresis. The gel is then immersed in alkali in order to separate DNA into single strands. DNA pattern transferred from gel to nylon membrane using Southern blotting - Hybridisation
Radioactive or fluorescent DNA probes are added (that are complementary to a known DNA sequence) and bind to complementary strands of DNA under particular conditions of pH and temperature. These DNA strands identify microsatellite regions as they are more varied than minisatellite regions. The excess probes are washed off - Seeing the evidence
X-ray images or UV light gives a pattern of bars which are unique to everyone except identical twins. This is a DNA profile
10
Q
PCR
A
A version of the natural process in which DNA is replicated, so allows scientists to produce a lot of DNA from a small original sample
1. Separating the strands
- temperature in PCR increased to 90-95°C for 30 seconds
- this denatures the DNA by breaking the hydrogen bonds holding the DNA strands together so they separate
2. Annealing the primers
- the temperature is decreased from 55-60°C and the primers bind (anneal) to the ends of the DNA strands
- the primers are required for the replication of strands to occur
3. Synthesis of DNA
- temperature increases to 72-75°C for one minute, as it 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. Taq polymerase is used, obtained from thermophilic bacteria
11
Q
uses of DNA profiling
A
- forensic science in criminal investigations. DNA left at a crime scene matched to suspects/criminal databases
- prove paternity of child when in doubt or in immigration cases
- identifying species to which an organism belongs
- identifying people who are at risk of developing particular diseases as certain microsatellite patterns can be linked to an increased incidence of particular diseases such as heart disease
12
Q
origins of DNA sequencing
A
- Frederick Sanger developed techniques for sequencing nucleic acids from viruses and then bacteria
- it involved radioactive labelling of bases and then gel electrophoresis
- Sanger sequencing can read 500-800 bases at a time and has gone on to sequence whole genomes
12
Q
The human genome project (HGP)
A
An international project in which scientists from a number of different countries worked to map the entire human genome, making the data freely available to scientists all over the world
- once a genome has been assembled, scientists identify the parts of a genome that code for specific characteristics or regions that are linked to a particular disease
13
Q
capillary sequencing
A
- where DNA sequences are separated by length in minute capillary tubes (line gel electrophoresis)
14
Q
next generation sequencing
A
- Sanger sequencing can be difficult and time consuming even for small sequences of DNA
- technological developments have led to new automated sequencing processes
- sequencing reaction takes place on a plastic slide called a flow cell (instead of electrophoresis)
- millions of fragments of DNA are attached to the slide and are replicated
using PCR - the replication produces clusters of identical DNA fragments that have the same coloured terminator
- all of the clusters are sequenced and imaged at the same time (massively parallel sequencing)
- it is integrated with computer technology which is highly efficient, so can sequence genomes quickly
15
Q
terminator bases
A
- modified versions of the 4 nucleotide bases that stop DNA synthesis when they are included
- the terminator bases are given different colour tags
- A is green, G is yellow, T is red, C is blue
- for example, an A base terminator would stop DNA synthesis at the location an A base would be added.
16
Q
The process of DNA sequencing
A
- the DNA strand is cut into fragments and mixed with primer, bases, DNA polymerase and terminator bases
- the mixture is placed into a thermal cycler which separates DNA strands at 96°C and anneals primer to strands at 50°C
- at 60°C, DNA polymerase adds complementary bases to create a new DNA strand
- terminator bases are added at random, ending the strands
- after many cycles, all possible DNA strands are produced and then separated by size using capillary sequencing
- lasers detect the colour of the fluorescent tags that correspond to the terminator base. This therefore determines the sequence of bases
- DNA analysis gives the correct DNA sequence (it produces a complementary strand)
17
Q
Bioinformatics
A
- the developments of the software, computing and statistical tests needed to organise and analyse raw biological data.
- used to generate data on RNA and DNA sequences and the relationship between genotype and phenotype
18
Q
Computational biology
A
- the use of data from bioinformatics to build theoretical models of biological systems
- can be used to predict what will happen in different circumstances
- important in the analysis of data from sequencing genomes to studying evolutionary relationships, epidemiology and structures of proteins
- high powered computers are used to make databases
19
Q
genomics
A
applying DNA sequencing methods and computational biology to analyse the structure and function of genomes
20
Q
why analyse the genomes of pathogens
A
- find out the source of an infection
- identify antibiotic-resistant strains of bacteria so antibiotics are only used when effective and making it easier to track the spread
- track the progress of an outbreak
- identify genetic markers for use in vaccines and regions of the genome that may be useful to target when developing new drugs
21
Q
DNA barcoding / International Barcode of Life project (IBoL)
A
- scientists use relatively short sections of DNA from a conserved region of the genome to identify species
- the section of DNA is small enough to be sequenced cheaply, yet varies enough to give clear differences between species
- it is easier than traditional methods of observation
- scientists have not yet come up with suitable regions to classify fungi and bacteria
22
Q
searching for evolutionary relationships
A
- DNA sequences of different organisms can be compared
- the base mutation rate of DNA can be calculated, so scientists can calculate how long ago two species diverged from a common ancestor
- scientists can build more accurate evolutionary trees
23
Q
proteomics
A
the study and amino acid sequencing of an organism’s full range of proteins produced by the genome (protein complement)
- the DNA sequence of the genome should in theory enable scientists to predict the sequence of amino acids and therefore the proteins it produces, but this is not always true
24
Why are the sequence of amino acids produced by an organism not always what would be predicted from the genome alone
Spliceosomes:
- 'pre-mRNA' is modified before it is translated on the ribosome
- exons to be translated are joined by spliceosome enzymes in a variety of ways, creating several versions of mRNA and therefore giving different proteins which result in different phenotypes
Protein modification:
- a protein coded by a gene may remain intact or be shortened or lengthened by other proteins to give a variety of other proteins
25
Synthetic biology
- an emerging area of research
- design and construct of artificial biological pathways, organisms or devices
- the redesign of existing biological systems
26
The process of genetically engineering a bacteria to produce human insulin
- mRNA is extracted from human pancreas cells
- a plasmid is extracted from bacteria to use as a vector
- mRNA is treated with reverse transcriptase to make complementary DNA (cDNA)
- the plasmid is cut with a restriction enzyme
- the plasmid and cDNA is fused using DNA ligase
- the recombinant plasmid is introduced into host bacterial cells
- the bacteria multiply in a fermenter and produce insulin
- the insulin is separated and purified to be used by diabetic patients
27
How are restriction endonucleases used in genetic engineering
- used to cut the required gene from the DNA and to cut the plasmid
- each type of restriction endonuclease cuts the DNA at specific base pairs
- the enzyme cuts the strands unevenly so there are 'sticky ends'
- these exposed unpaired base pairs make it easier to insert the desired gene into the DNA of a specific organism
28
How are bacterial plasmids specialised to be used as vectors in genetic engineering
- often chosen because they contain two marker genes, for example a gene for fluorescence and antibiotic resistance
- this marker genes enable scientists to determine if the bacteria have taken up a plasmid for example by growing the bacteria in media containing the antibiotic
- the DNA fragment that manufactures the desired protein is joined within the second marker gene
- if the DNA fragment is inserted successfully the marker gene will not function , for example the bacteria will not emit fluorescence
29
Transformation by chemical treatment
-culture the bacteria cells and plasmids in a calcium rich solution and increase the temperature
- causes the bacterial membrane to become permeable so the plasmids can enter
30
Transformation by electroporation
- a small electrical current is applied to the bacteria making the membranes very porous so the plasmids can enter
- can also be used to move DNA fragments directly into the nucleus of a eukaryotic membrane, but the electrical current has to be carefully controlled
31
transformation
the process of transferring the plasmid with the recombinant DNA into the host cell
32
electrofusion
- tiny electric currents are applied to the membranes of two different cells
- this fuses the cell and nuclear membranes of the two cells together to form a hybrid/polypoid cell containing DNA from both
- used to produce GM plants
- electrofusion is used differently and less successfully with animal cells, and polypoid cells rarely survive in living organisms
- important in producing monoclonal antibodies which are used to identify pathogens and treat diseases
33
genetic engineering in prokayotes
- bacteria and other organisms are used to produce substances useful to people such as insulin and other hormones, clotting factors for haemophiliacs, antibiotics, vaccines and enzymes used in industry
- much easier to do than with eukaryotes
34
method for genetic engineering in plants
- use a bacterium that causes tumours in healthy plants
- cut leaf to allow bacteria to 'infect' plant cells
- expose leaf to bacteria containing genes for weedkiller and antibiotic resistance
- allows bacteria to deliver genes to leaf cells
- expose leaf to antibiotics to kill unmodified leaves
- surviving cells multiply to form a callus
- allow callus to produce roots and shoots
- plants transferred to soil to develop into adult plants that are resistant to weedkiller (glyphosphate)
35
genetic engineering in animals
- harder to genetically modify than plants
- especially hard to engineer mammals
- animal cells membranes are less easy to manipulate than plant cell membranes
- important technique to enable animals to produce medically important proteins and to try and cure human genetic diseases such as CF and Huntington's
36
What characteristics are given to genetically modified plants?
- pest resistance
- disease resistance
- herbicide resistance
- extended shelf life
- nutritional value
- medical uses like producing vaccines
- adapted to certain growing conditions
37
perceived risks of genetically modified plants
- resistance could spread to wild populations of plants or bacteria, creating superweeds
- reduced genetic diversity
- introduces a selection pressure for pests
- patenting GM crops cam make seeds expensive and infertile, disadvantaging people in less economically developed countries
38
somatic gene therapy
- replacement of faulty gene in nuclei of body cells with a healthy allele
- must be done in many cells
- the healthy allele is not passed on to offspring
- it does not cure genetic disease
39
germ line gene therapy
- replacement of faulty gene in nuclei gamete or early embryo with a healthy gene
- Only needs to be done in one or two cells but required for every child born to an affected individual
- healthy allele will function in individual and pass onto children
- cures the genetic disease
40
Pharming
Creating animal models:
- the addition or removal of genes so that animals develop certain diseases
- animals act as models for the development of new therapies
Creating human proteins:
- introduction of a human gene coding for a medically required protein
- animals sometimes used when the proteins needed is too complex for bacteria to produce
- human gene introduced into fertilised animal egg
- often introduced into sheep, cow or goat egg along with a promoter sequence so the gene is only expressed in the mammary gland
- the animal then produces milk with the desired protein in when it matures
