Manipulating genomes Flashcards
DNA sequencing
technique that allows genes to be isolated and read
Fred Sangers method of DNA sequencing
- DNA cloning- desired gene isolated by restriction enzymes, then inserted into a plasmid so when DNA divides, multiple copies of the gene are produced
- Made single stranded- heat at 94-96°C
- Sequencing
-single strand placed in one of 4 dishes containing nucleotide bases, DNA polymerase, DNA primers and radioactively labelled base (either A T G or C)
-normal bases pair with complementary base on single strand
-pairing is terminated when a radioactively labelled base is paired
-this means the end of each fragment has a radioactively labelled base
-DNA fragments passed through a gel via electrophoresis
-this sorts fragments by length, shorter fragments travelled further
DNA sequencing machine
-uses fluorescent dye instead of radioactive base
-dyes glow when scanned with a laser
-the light is identified by a computer
-this method requires technicians who can read autoradiograms
Pyrosequencing
-long length of DNA to be sequenced is cut into fragments by a nebuliser
-these fragments are then degraded into single stranded DNA and immobilised
-a primer is added and DNA is incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase, APS and luciferin
-only one of the 4 activated nucleotides are added (ATP GTP CTP or TTP)(activated as have 2 extra phosphate groups)
-the activated base that is complementary to the strand will be incorporated into
-this releases the 2 extra phosphoryls as pyrophosphate
-in the presence of APS, ATP sulfurylase converts pyrophosphate to ATP
-in the presence of ATP, luciferase converts luciferin to oxyluciferin which generates visible light
-the amount of light generated is proportional to the amount of complementary activated nucleotides that are added
Bioinformatics
where the data generated is stored from DNA sequencing
3 types of genome wide comparisons we make
(applications of sequencing)
-comparisons between species
-evolutionary relationships
-variation between individuals
Comparisons between species
-human genome compared to other species, found few genes were unique to us
-verifies that genes that work well are conserved by evolution
eg. pig and human gene for insulin is similar
Evolutionary relationships
-compare the genome of organisms thought to be related
-DNA from bones and teeth of extinct animals can be sequenced so we can verify their evolutionary history
Variation between individuals
-all humans are genetically similar apart from rare occasions where mutations occur
-the places where the mutations occur are called polymorphisms or SNPs
-methylation- plays a role in regulating gene expression + used to understand the development of diseases via epigenetics eg. cancer.
Predicting amino acid sequences of proteins
-need sequenced genome
-know which part of the gene codes for the protein
-they can then determine the primary structure of the protein
-need to know which sections of the gene are introns and exons
Synthetic biology
-concerned with designing and building useful biological devices and systems
eg. info storage, production of medicines, nanotechnology
DNA profiling procedure
-DNA is obtained from the individual by salvia, hair or bone etc
-the DNA is cut by restriction enzymes at 13 specific points. These points are where the short tandem repeats are, which vary in length between individuals
-the fragments of STR are separated by gel electrophoresis, where the larger fragments travel the shortest distance
-then a banding pattern can be seen
-use this to compare DNA to another persons and find how closely related they are, by seeing how many of their STRs are the same
Applications of DNA profiling
-identify nazi war criminals hiding in south america
-identify victims after plane crashes/ terrorist attacks etc
Polymerase chain reaction and what it relies on
-where a short length of DNA is amplified to form lots of copies
Relies
-DNA being antiparallel
-each strand having a 5’ and 3’ end
-DNA only grows in the 5’ to 3’ direction
-base pairs pair with complementary bases
How PCR differs from DNA replication
-only uses short sequences
-requires DNA primer
-cycle of heating and cooling
Stages of PCR
-DNA mixed with nucleotides, primer, Mg2+ and Taq DNA polymerase
-heated to 94-96°c to make single stranded
-then cooled to 68°c so primer can bind
-then heated to 72°c as optimum for Taq DNA polymerase
-Taq DNA polymerase catalyses the addition of nucleotides in the 5’ to 3’ direction
-when it reaches the end a whole new double strand has been created
-the process continues exponentially- 1,4,9,25,35
-to calculate number produced= 2^number of cycles
-to calculate the number of cycles= log2(number produced)
Applications of PCR
-tissue typing- reduce rejection after transplant
-detect mutations
-identify viral infections- as can detect small amount of viral genome amongst host cell DNA eg HIV
-forensic science- identify criminals
Electrophoresis
process used to separate proteins or DNA fragments of different sizes
Electrophoresis method
-DNA cut by restriction enzymes at 35-40°C
-tank is set up with agarose gel and buffer solution
-loading dye is added to tubes containing digested DNA
-the DNA and loading dye is added to the wells in the gel
-electrodes are added and connected to a battery. The negative electrode is at the end with the wells as the negative phosphates on DNA will be attracted to the positive electrode, and therefore will move through the gel
-fragments move at different speeds, smaller travel faster
-buffer solution is poured away and a dye added which stains DNA
Separating proteins by electrophoresis and the uses
-same as separating DNA but detergent eg. sodium dodecyl sulphate (SDS) is used
-this equalises the surface charge on the molecules, so proteins can separate according to molecular mass
-if SDS isn’t used, proteins can be separated by surface charge
Uses
-diagnosis of sickle cell anaemia and leukaemia etc.
DNA probes and uses
-short single stranded length of DNA that is complementary to the section of DNA being investigated
-labelled using a radioactive or fluorescent marker
Uses
-locating a specific gene needed for genetic engineering
-genome comparison studies
-identify an allele for genetic disease
Microarrays
-number of different probes on a fixed surface
-DNA complementary to the microarrays will anneal
-a reference marker is used first to check its working and then the test subject is added
Genetic engineering
genes are isolated from one organism and inserted into another using suitable vectors
Stages of genetic engineering
-requieres gene obtained by restriction enzymes
-copy of gene place inside vector eg. plasmid
-the vector carries the gene into recipient cell
-the recipient expresses the novel gene
Ways of obtaining required gene
-use DNA probe to find gene and cut with restriction enzymes
-synthesise gene using an automated polynucleotide synthesiser
-if already know sequence they can use PCR to amplify DNA
-obtain mRNA and use reverse transcriptase to form single strand of complementary DNA, then use PCR to amplify
Different vectors and how genes can be placed
-use plasmid which is cut by restriction enzymes, forming sticky ends
-use a virus which will carry it to host cell
Methods of getting vector into recipient cell
-heat shock treatment- bacteria heated and cooled in presence CaCl2. This makes membrane more porous and allows recombinant vector in
-electroporation- high voltage pulse applied which disrupts membrane
-electrofusion- use of electrical fields to introduce DNA to cells
-transfection- package DNA into bacteria which infects host cell
Direct method of introducing gene to recipient
Gene gun- small pieces of gold or tungsten are coated with DNA and shot into plant cells
Reverse transcriptase
-catalyses formation of cDNA (complementary DNA) using RNA template
-this is the reverse of transcription
Obtaining insulin from genetically modified bacteria
-add reverse transcriptase and to form cDNA, treat with DNA polymerase to make double stranded
-unpaired nucleotides at the ends form sticky ends
-ligase sticks insulin gene into plasmids from E. coli
-E coli are mixed with recombinant plasmids under heat shock treatment with CaCl2 so they take up plasmids
Tests to know if bacteria has insulin gene inserted
-place on agar plate of ampicillin. If bacteria survive, the plasmid must be inserted as it contains the ampicillin resistant gene
-next place on agar plate of tetracycline. If the bacteria survives, then the insulin gene isn’t inserted in the plasmid as the insulin gene blocks the tetracycline resistant gene, making it no longer resistant.
Principle of gene therapy
-insert a functional allele of a particular gene into cells that contain a non functioning allele
-so when functional allele is expressed, the individual will produce the functioning protein and no longer have the symptoms of the disorder
Somatic gene therapy and use to treat genetic diseases
-gene therapy by inserting functional alleles into body cells
Treatment cystic fibrosis:
-inhale aerosol containing liposomes with CFTR alleles
-liposomes pass through the plasma membrane and then nuclear envelope
-allele inserts itself into host genome
-mRNA is transcribed from the CFTR allele and then translated
-this forms a normal chloride channel, removing the usual symptoms
Vectors used in gene therapy
-liposomes- sphere of lipid bilayer containing the functional allele
-viruses- modified so don’t cause infection and then enter cells taking the functional allele with it. Problems eg. may provoke immune response, may become immune to virus, may insert allele at wrong place so risk of cancer
-artificial chromosomes- insert gene into artificial chromosome that would co exist with other 46 chromosomes
Germ line therapy
-altering the genome of gametes or zygotes
-not only the cells of the individual will be altered, but also their offspring
Problems
-may disrupt the expression or regulation of other genes increasing risk of cancer
-ethical issues as it changes genetic material of original patient and therefore changes genetic makeup of the descendants who wouldn’t have given consent
