6.3 Manipulating Genomes Flashcards
What is DNA sequencing?
A technique that allows genes to be isolated and read, to determine the sequence of bases.
Outline Sangers approach
- Extract DNA cut into fragments using restriction enzymes, degrade into single strands.
- Amplify by PCR
- Place into 4 separate dishes
- each dish contained a solution with four terminator bases A, C, T, G, plus DNA polymerase, primers and free DNA nucleotides - If a modified base was added into the synthesized complementary strand of DNA, no more bases could be added
- Fragments of varying length passed through a gel by electrophoresis -> sorted into lengths
- nucleotide base at the end of each fragment was read according to its radioactive label
- Place sections in order by matching overlapping regions
Outline high throughput/ pyrosequencing
- DNA cut into fragments using a nebuliser, and degraded into single strands.
- sequencing primer is added
- only one of the four activated nucleotides (ATP, TTP, CTP, GTP) is added at any one time and any light generated is detected
- one activated nucleotide is incorporated into a complementary strand of DNA (help of DNA polymerase)
- two extra phosphate groups are released
- in presence of APS, ATP sulfurylase converts the pyrophosphate to ATP
- luciferase converts luciferin to oxyluciferin which generates visible light that can be detected by a camera
What is bioinformatics? How is it used?
Large amount of universal data on DNA and proteins.
Access to amino acid sequences
Can compare sequences with newly sequenced alleles
Allows scientists to make comparisons with the genomes of other organisms using the databases available.
Use computational biology for modelling of protein structures from DNA base sequences
Epidemiology:
What are the applications for gene sequencing?
1) comparisons between species, e.g. found only a few genes unique to humans.
comparing genomes of organisms thought to be closely related helps to confirm their evolutionary relationships. DNA from bones and teeth of some extinct animals can be amplified and sequenced.
2) allows for comparisons between individuals: although humans all have the same genes (mostly), our alleles differ due to mutations, so we can compare these genotypes to phenotypes.
3) predicting amino acid sequences of proteins (closely linked to bioinformatics and computer modelling)
4) synthetic biology (designing and building useful biological devices and systems) -> e.g. production of drugs by genetically engineering bacteria or yeast, novel proteins, biosensors, and materials produced for nanotechnology.
What is the DNA profiling procedure?
- DNA obtained
- DNA digested with restriction enzymes, that cut the DNA at specific recognition sites. These will cut the DNA into fragments which vary in length between individuals
- Fragments separated by gel electrophoresis and stained. Larger fragments travel the shortest distance
- Banding pattern can be seen
- DNA to which the individual is being compared is treated with the SAME restriction enzyme and also done in electrophoresis.
- The banding patterns of the DNA can then be compared
What part of the DNA is analyzed?
Short tandem repeat sequences (in introns). These contain mini satellites and micro satellites
Highly variable short repeating lengths of DNA that are polymorphic, and the NUMBER varies from person to person (not the code)
What are the applications of DNA profiling?
- Forensic science: identify war criminals, establish innocent suspects, identify victims body parts after disasters, identify remains
- Maternity and paternity disputes: comparing DNA profiles of mother, father and child can establish maternity or paternity
- Analysis of disease: protein electrophoresis can detect type of haemoglobin present and aid diagnosis of sickle cell, as well as detect a varying number of repeat sequences for conditions such as Huntingtons.
What is the purpose of PCR?
biomedical technology that can amplify short lengths of DNA to thousands of millions of copies
Explain the denaturation step. Why?
Mixture is heated -> denatures DNA to break hydrogen bonds between two strands, to separate them, to expose the nucleotide bases.
Explain the annealing steps. Why are primers needed?
- Mixture cooled to around 68 degrees celsius so the primers can anneal to one end of each single strand of DNA
- Taq polymerase can now bind to end where there is double stranded DNA
Explain the extension steps. Why is the temp raised?
- Temperature raised to 72 degrees to keep the DNA as single strands and this is the optimum temperature for Taq polymerase
- Taq catalyses addition of DNA nucleotides to single-stranded DNA molecules, starting at the end with primer and proceeding in 5’ to 3’ direction.
- Taq reaches the other end of the DNA molecule, then a new double strand of DNA has been generated.
How do you calculate the number of DNA molecules after n cycles of PCR?
2^n
Compare and contrast DNA replication with PCR
Similarities:
- polymerases
-complementary base pairing
- breaking and forming of hydrogen bonds
- phosphodiester bond formation
Differences:
- Taq vs DNA
- needs primers for PCR
- PCR needs cycle of heating/cooling
- only short sequences in PCR, vs whole DNA molecule in replication
Explain why PCR is used in DNA fingerprinting
- possible to amplify small quantities
- relatively short time
- make exact copies
Why is Taq polymerase chosen?
- thermostable
- so PCR can be cycled repeatedly without stopping
What are concerns people have about GM of bacteria?
antibiotic resistance
What are some applications of PCR? (7)
Used to amplify DNA samples for many applications:
Tissue typing: reduce risk of rejection of transplant
Detection of oncogenes: medication can be tailored to patients
Detecting mutations: DNA analysed for presence of mutation that leads to genetic disease
Identifying viral infections
Monitoring spread of infectious disease
Forensic science -> amplified for DNA profiling for paternity tests or criminal identification
Research: analysis of DNA from extinct ancient sources for example
How is electrophoresis used for DNA profiling?
process used to separate proteins or DNA fragments of different sizes
when an electric current is applied, the DNA fragments move towards the anode (positive electrode) since DNA has negatively charged phosphate groups
smaller, shorter fragments have less mass so will travel further quicker
Describe electrophoresis more in depth (equipment used, why, etc)
Create an agarose gel plate in a tank. Wells (a series of groves) are cut into the gel at one end
Submerge the gel in an alkaline solution /buffer -> Solution carries charge to separate fragments✓
Load (insert) the fragments into the wells using a micropipette
The fragments are not visible. What is done about this?
(use) Southern blotting / described
AND to transfer fragments to a
membrane e.g adsorbent paper or nitrocellulose✓
What are DNA probes?
Single stranded sequences of DNA complementary to section of DNA being investigated.
They may be labelled using: radioactive marker (once probe has annealed, it can be revealed by exposure to photographic film), OR fluorescent marker that emits colour on exposure to UV light.
to , visualise / AW , bands/
patterns ✓
How is separating proteins different? What is used?
idea that different proteins have
different overall charges (1)
idea that (binding of) SDS makes
all proteins negatively charged (1)
idea that proteins will be separated
by, mass / length (1)
idea that proteins move in the
same direction (1)
Can be used to analyse types of haemoglobin in diagnosis of diseases such as sickle cell, thalassaemia and leukaemia.
What is a microarray?
- a fixed surface, where scientists can place a number of diff probes on it, to detect mutated alleles that cause genetic diseases for example
- test and reference DNA labelled with fluorescent markers. where a test subject and a reference marker both bind to probe, the scan reveals fluoresence of both colours, indiciating that particular sequence in the test DNA.
How do you obtain the required gene for genetic engineering?
- mRNA can be obtained - reverse transcriptase catalyses formation of single strand of cDNA. add primers and DNA polymerase = double stranded DNA.
- DNA probe locates a gene within the genome and the gene can be cut out using restriction enzymes
Summarise the process of placing the gene into a vector
- plasmids can be obtained from bacteria and mixed with the SAME restriction enzymes that cut the plasmid at specific recognition sites
- the cut plasmid has sticky ends which are complementary to those of the gene. nucleotide bases can be added to the ends of the gene to be inserted if needed.
- the cut plasmid should anneal. DNA ligase enzyme catalyses this, via condensation reactions
- OR a gene may be sealed into a weakened virus that could carry it into a host cell
How do you get the vector into the recipient cell?
- heat shock treatment with calcium chloride, where ca2+ surround the negatively charged parts of DNA and phospholipids, reducing repulsion between foreign DNA and host membranes = bacteria walls and membranes become more porous and allow recombinant vector in
- electroporation: high voltage pulse
- electrofusion: electrical fields
- transfection: packaged into bacteriophage which transfect host cell
- directly using gene gun (gold or tungsten coated with the DNA) for plants
What sequences do restriction enzymes always recognise? What ends do they produce? What is their co factor?
palindromic
can be sticky (staggered cut) or blunt ends
magnesium ions
Describe the steps in the production of insulin from GM bacteria. How can we identify if plasmids have been taken up?
- Obtain mRNA from beta cells of islet of Langerhans
- Add reverse transcriptase enzyme and DNA polymerase -> double strand of gene
- Produce sticky ends by adding free unpaired nucleotides
- Use ligase to insert insulin gene into plasmids from E.coli -> recombinant plasmids
- E coli bacteria mixed with plasmids and subjected to heat shock, so they take them up
- Cultured in large numbers
Can use antibiotic resistance marker genes
Advantages vs disadvantages of GM microorganisms such as E.coli?
Can make human insulin to treat diabetics
Microorganisms could escape and transfer marker genes for resistance to others, however Gm bacteria are also modified so they cannot live outside the lab.
Advantages vs disadvantages of GM plants e.g. Bt tobacco and maize?
- GM plants can now produce Bt toxin which is toxic to insects; eliminate to spray it around and possibly contaminate
- Toxic to monarch butterflies, but they dont take nectar from the plants in the wild.
Advantages vs disadvantages of soya beans GM?
resistant to herbicide, so competing weeds killed
- production of ‘superweeds’
Advantages vs disadvantages of golden rice
contain beta carotene producing gene, to prevent vitamin A deficiency
some were concerned about buying them constantly, but free licences have been issued
Advantages vs disadvantages of Gm pathogens
viruses modified to have no virulence can be used to make vaccines
problems with virus use in gene therapy, as allele could be inserted problematically
Advantages vs diasdvantages of pharmaceutical proteins
genes can be inserted into goats or sheep, and protein harvested in milk
concerns for welfare, however they’re likely to be well looked after
Describe somatic gene therapy
inserting functional alleles into body cells
alterations made are not passed to offspring
short term
How is gene therapy used to treat cystic fibrosis?
- Aerosol containing liposomes with CFTR alleles
- Liposome passes through plasma membrane
- Functioning allele transcribed into mRNA, and translated into normal chloride ion channels
Has to be repeated at regular intervals though
Viruses have been used as vectors. What are the problems?
- may still provoke inflammatory response
- may become immune to virus
- may insert allele in a disruptive way for gene regulation or at increased risk of cancer
Describe germ line gene therapy
inserting functional alleles into gametes or zygotes
offspring may inherit the alleles too
long term
Explain how DNA sequencing allows the sequence of amino acids in a polypeptide to be predicted (2)
sequence / order , of bases codes
for , sequence / order , of amino
acids ✓
(each) triplet / three bases / codon
, (codes) for , one amino acid ✓
Suggest how the interdisciplinary field of bioinformatics may be useful in determining whether a newly sequenced allele causes a genetic disease (2)
base sequence of normal allele
and (known) alternatives held (in
database) (1)
computational analysis allows
rapid comparison of sequences
with newly sequenced allele (1)
amino acid sequence / protein
structures, also held (in database)
(1)
idea of
computer modelling of new protein
structure from base sequence (1)
Explain how DNA sequencing can help scientists understand how the West Highland terriers genes affect its probability of devevloping CPF (2)
idea of compare genomes of, dog
breeds / individual dogs ✓
idea of identify, alleles / genotypes /
base sequences (in WHTs), that
are present (only) in dogs with CPF
✓
idea of identify dogs that are
carrying (the allele for) CPF ✓
(use of) computational biology /
bioinformatics, to link genes with
CPF ✓
idea of linking DNA sequences to
specific proteins (i.e. proteomics) ✓
Suggest why proteins are heated before being placed in electrophoresis gel (1)
denature / unfold, protein AND
idea of exposes charges or
hydrophobic region (1)
Statement ‘GM plants and animals should be classed as new species’. Outline one experiment or investigation that would provide evidence to support or contradict the statement (3)
Fertility
breed GM stock with non-modified
stock (1)
see if offspring fertile (1)
if so they should be classed as the
same species (1) ora
Morphology
Compare several individuals from
GM and non-GM groups (1)
in respect of several physical
structures (1)
if similar they should be classed as
one species (1) ora
Genetics
compare DNA (1)
by electrophoresis (1)
same pattern should be classed as
one species (1) ora
Explain how inserting a new gene into a chromosome could affect the functioning of other genes in that chromosome (2)
frameshift ✓
altered triplet(s) ✓
adjacent / nearby, genes (on same
chromosome) switched, on / off ✓
idea that new gene could disable a
functioning gene if inserted into it ✓
