one region of the chromosome is flipped and reinserted
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duplication mutation
region of the chromosome is repeated, resulting in increase dosage from genes in gene in regions?
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translocation mutations
region from one chromosome is aberrantly attached to another chromosome
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transcriptional factors
= simulate the region of DNA to begin transcription
binds to specific region of the DNA
can be blocked by inhibitor, DNA is not transcribed, gene is not expressed
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oestrogen
steroid hormone, lipid soluble molecule
combine with receptor/transcription factor, changing its shape so inhibitor is released and DNA binding site is exposed; will now bind to promoter region of DNA, initiating transcription
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totipotent
can divide and specialise into any type of body cell and the placenta
early stage of fertilised eggs/mammalian embryonic stage only
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pluripotent
can specialise into any body cell, but not cells that make up the placenta
divide in unlimited numbers and used in treating human disorders
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multipotent
differentiate into a few/limited number of different types of cells
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unipotent
can only differentiate into a single type of cell eg. cardiomyocytes of the heart
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induced pluripotent stem cells
pluripotent stem cells produced from unipotent stem cells (adult somatic cells)
they are genetically altered in lab by including genes and protein transcription factors to express themselves
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how do totipotent cells initiate specialisation?
part of DNA is translated
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translation factors: small interfering RNA (siRNA)
double stranded RNA
1. cut down to smaller pieces by dicer 2. RISC complex is formed and binds to mRNA, degrading it
1. forms RISC complex 2. binds to mRNA that is complementary to RISC
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how translation is stopped, when mRNA binds to RISC
mRNA is hydrolysed by enzyme - RNA hydrolase
or, ribosomes are prevented from attaching to mRNA
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epigenetics
= heritable change in gene function without changes to base sequence of DNA
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how epigenetics work?
histones and DNA are covered in tags eg. methyl
forming second layer called epigenome, determines shape of DNA-histone complex
DNA code is fixed, however, epigenome is flexible to change by environment
DNA is exposed = expressed gene
tightly packed histones = not expressed gene
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acetylation
bind to = (amino acid of) histones
effect on DNA = (more acetyl) loosely packed histones
effect on gene expression = expressed
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methylation
bind to = (cytosine) DNA base
effect on DNA = (more methyl) tightly packed histones
effect on gene expression = not expressed
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epigenetic treating disease
= epigenetic changes are reversible
eg. drugs can inhibit enzymes involved in DNA methylation
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tumour
= uncontrolled cell division
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difference between benign and malignant tumours
benign tumours are localised to one area of the body, whereas, malignant has effects across whole body as it can spread
benign does not cause cancer; malignant causes cancer
benign grow slowly; malignant grow rapidly
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how do tumours cause harm?
damage organ concerned
cause blockage/obstruction
damage/exerting pressure on other organs
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tumour suppressor gene function
codes for proteins that slow down cell division, repair mistakes in DNA, and tells cells to die
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hypermethylation of **tumour suppressor gene**
1. hypermethylation occurs in specific, promoter region of TSG 2. histones become tightly packed and transcription factors cannot reach DNA, TSG is not expressed 3. stops transcription of TSG 4. **proteins** that slow down cell division not produced/translated 5. leading to increase of/uncontrolled cell division
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proto-oncogenes
= stimulate a cell to divide when growth factors attach to a protein receptor on its cell surface membrane
activates DNA to replicate + cell to divide
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hypermethylation of **oncogenes**
growth factors are produced in excess
permanent activation of proto-oncogenes
cell divides too rapidly and out of control
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increased oestrogen concentration on proto-oncogenes
cause proto-oncogenes of cells in breast to develop into oncogenes
ie. oestrogen releases inhibitor, activating genes so uncontrolled cell division
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genome project (spec)
sequencing projects have read the genomes of a wide range of organisms
determining the genome of simpler organisms allows the sequences of proteins that derive from genetic code (proteome) of organisms to be determined, applications such as identification of potential antigens for use in vaccination
in more complex organisms, presence of non-coding DNA and of regulatory genes means knowledge of the genome cannot be easily translated into proteome
sequencing methods are continuously updated and have become automated
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recombinant DNA technology
= involves transfer of fragments of DNA from one organism/species to another
genetic code is universal, so translation and transcription are too
transferred DNA will be translated within recipient (transgenic) cells
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producing DNA fragments
1. reverse transcriptase: conversion of mRNA to cDNA (complementary DNA) 2. restriction endonuclease: cut a fragment containing the desired gene from DNA 3. gene machine: create the gene
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using reverse transcriptase
take cell that rapidly produces required protein
cells have large quantity of relevant mRNA which is extracted
reverse transcriptase (+ DNA polymerase) used to make cDNA
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using restriction endonuclease
= cuts up the viral DNA
cuts at recognition site
producing sticky ends, known as palindromic sequence
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using gene machine
determine nucleotide sequence for desired protein
use machine to produce gene with no introns and duplicated by PCR
advantage: very accurate in short time and free from introns (so prokaryotes can translate)
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plasmid
= small circular piece of DNA found naturally in bacteria
separate from main bacterial DNA
contains only a few genes
have the ability to insert themselves into bacteria
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vector
= carries gene from one organism to another
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why use bacteria as recipient cell?
= small and easy to manipulate
reproduce quickly
have plasmids which can be used as vectors
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in vi**v**o: function and process
function = to clone DNA (in organism/bacteria)
processes = restriction endonuclease and ligase used to insert a gene into vectors, which are then transferred to host cells
genetic markers are used to identify transformed cells
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in vi**v**o cloning
1. restriction endonuclease used to cut desired gene 2. cuts at recognition site; both plasmid and gene 3. fragment of DNA has promoter and terminator region added to it 4. promoter region attaches RNA polymerase and transcription factor; terminator releases RNA polymerase (transcription starts and ends) 5. DNA ligase inserts foreign DNA into plasmid by catalysing the formation of phosphodiester bonds between gene and plasmid 6. plasmid is mixed with bacteria: cold calcium chloride used to shock bacteria so it takes up plasmid (known as transformation)
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in vi**v**o cloning: why are there more than one transformations of plasmid/gene
all cut DNA have the same complementary sticky ends/base sequence
process of sticky ends joining is random
so, may form plasmid + gene, plasmid + no gene, and no plasmid + gene
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in vi**v**o cloning: replica plating
circular, velvet sterile pad pressed on agar plate
bacteria stick to it so it can be transferred to another plate
pressed onto new plate so colonies will grow in exactly same position
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in vi**v**o cloning: industrial
on large scale:
must contain all nutrients for growth and replication;
supply oxygen;
temperature and pH must be controlled for optimum growing conditions
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in vi**tr**o cloning: function and process
function = to clone DNA (in test tube) (aka DNA amplification)
processes = heating to separate DNA strands
cooling - allow primers to attach
heating DNA polymerase binds free nucleotides
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in vi**tr**o cloning: PCR
polymerase chain reaction
1. DNA heated to 90-95°C 2. DNA is denature and strands separate 3. cooled to temperatures below 70°C 4. primers added (annealation) 5. (Taq) polymerase and nucleotides added and attach by complementary base pairing 6. increase temperature to 70-75°C 7. polymerase joins nucleotides together to form new strand 8. cycle repeated (20-30 times)
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primer
short length of double stranded DNA with complementary bases
= attach to starting point of gene, starting point for polymerase to begin the new DNA strand
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gene therapy: function and process
function = replace defective gene/treat genetic disease with (healthy) genes
process = use a vector (harmless virus or liposome) to carry gene into human cell
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DNA probes: function and process
function = to locate specific gene
process = probe is complementary to gene/part of gene of interest
probe mixed with single stranded DNA
probe binds to DNA (DNA hybridisation)
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DNA probe
= short piece of single stranded DNA, complementary to known base sequence/gene
radioactive probes identified by x-ray photographic film
fluorescent probes emit fluorescence/light under UV light
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DNA hybridisation
probe is complementary to part of gene of interest
DNA of interest is treated to separate the 2 strands
DNA is mixed with probe which binds to complementary bases
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genetic screening
for health risk, drug response, and heritable conditions
eg. notice mutation of a gene, family history of genetic disease
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genetic counselling
give advice and information for people to make decisions about themselves and offsprings (eg. the likelihood of the child being born with a disease)
research family history of inheritable disease
make people aware of further medical tests
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genetic fingerprinting: function and process
function = determines with genetic variability within a population
processes = DNA cuts using VNTRs and cloned by PCR
separated by gel electrophoresis
radioactive (or fluorescent) DNA probes are used to bind to core sequences to identify strands
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gel electrophoresis: function and process
function = (gel electrophoresis) separate DNA according to size/mass
process = fragments placed into a well at one end of agarose gel
electric current passes through the gel
pieces of DNA are attracted to positive charge
smaller section of DNA travel further
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how gel electrophoresis works?
DNA is negatively charged
VNTRs of DNA are cut by restriction endonuclease, after the quantity is increased by PCR
placed in the wells of agarose gel
current induced at negative electrode, repelling DNA to the positive DNA
smallest DNA travel furthest
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Southern Blotting
DNA separated into single strands by alkaline solution
thin nylon membrane is placed over the gel, after electrophoresis
draws gel containing DNA up, in to nylon membrane by capillary action: same exact place as on electrophoresis
DNA probes added to membrane so they attach to DNA: UV light used to view fluorescent probe, or X-ray film used to view radioactive label
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VNTRs
variable numbers tandem repeat
genome of organism contains many repetitive intron bases eg. GCGCGCGCGC
number and length of VNTRs are unique to everyone; more similar = more closely related
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gel electrophoresis (mark scheme)
1. DNA is cut by restriction endonuclease 2. use gel electrophoresis 3. separate according to mass/length 4. Southern Blotting (nylon membrane) 5. alkaline conditions (make single stranded) 6. apply probe 7. radioactive/fluorescent 8. reference to VNTRs 9. autoradiography
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DNA sequencing: function and process
function = determine the base sequence of a gene
process = automated process and continually updated