Topic 8 Flashcards
Isolating target gene stages
1) restriction enzymes
2) reverse transcription
3) artificial synthesis gene
Isolating target genes - restriction enzymes
- DNA contains palindromic sites
- restriction enzymes cut DNA at specific palindromic sites called restriction sites
- if theres a restriction site either side of target gene
- restriction enzymes can be use to cut it out
- using restriction enzymes leaves DNA with sticky ends
Isolating target genes - reverse transcriptase
- cells only have 2 copies of each gene (in the nucleus), it is hard to access
- enzyme that does transcription backwards
Isolating target genes - artificial synthesise gene
- use a ‘gene machine’ to make DNA from scratch
- join about 25 nucleotides together at once
- forms an oligonucleotide
- join oligonucleotides together to form a synthetic gene
- design your own gene
Inserting target gene stages
1) isolating target gene
2) insert gene into a vector
3) insert vector into bacteria
Inserting target genes - isolating target gene
- through a: gee machine, reverse transcriptase, restriction enzymes
- needed to add: a promoter region, terminator region, sticky ends, marker gene
Inserting target genes - insert gene into a vector
- use the same restriction enzymes to cut plasmid (sticky ends are complementary
- DNA ligase reform the phosphodiester bonds
- forms recombinant DNA
Vector
Something that’s used to move DNA from one place to another
Recombinant DNA
DNA from more than one source/ organism
Inserting target genes - insert vector into bacteria
- transgenic organism - contains recombinant DNA
- ice cold calcium chloride
- heat shock (increases permeability of bacterial cell wall)
Marker genes
Genes that are paired with target genes to check if the vector has been inserted properly
Process of marker genes
- vector are often not take up by bacteria
- to tell which bacteria transformed you need marker gene
- transformed bacteria contains the recombinant DNA (target + marker gene)
- Marker genes can be easily identified
- only bacteria that have accepted the vector (transgenic bacteria) are selected and cultured
UV fluorescence as marker genes
Will fluoresce under UV
Antibiotic resistance as a marker gene
Will be able to survive in a culture with antibiotic
What is the use of PCR
- used to amplify DNA
- sometimes called in vitro DNA amplification
What is needed for PCR
- DNA sample
- free DNA nucleotides
- primers (need to select which part of DNA is copied)
- DNA polymerase
What are primers
Short sequences of DNA that are complementary to the start of DNA sample
Steps of PCR
1) heat to 95C
2) cool to 50C
3) heat to 70C (allows rate of reaction to happen fast)
4) repeat
Why do you heat up to 95C for PCR
- break H bonds
- Make DNA single stranded
Why do you cool sample to 50C in PCR
- allows primers to bind
- complementary base pairing
- DNA double stranded
- DNA polymerase can bind
Why do you heat sample to 70C in PCR
- DNA polymerase adds complementary nucleotides
- forms phosphodiester bonds
Why do you repeat the PCR method
each cycle we double the DNA
Summary of gene technology stages
1) isolate target gene
2) insert gene into vector
3) insert vector into bacteria
4) identify transgenic organism
5) culture transgenic bacteria
6) extract + purify protein
What do you use to isolate target gene
- restriction enzymes
- (gene machine, reverse transcription, promoter + terminator)
What is used to insert a gee into a vector
- same restriction enzyme
- sticky ends to be complementary
- DNA ligase
What is used to insert vector into a bacteria
- recombinant DNA
- Ice cold CaCl2 + Heatshock
What is used to identify transgenic organisms
- marker gene
- U.V. Fluorescence/ radioactivity/ antibiotic resistance
What is used to culture transgenic bacteria
- transcribe + translate recombinant DNA
- Make the protein of target gene
Gene therapy
Changing faulty alleles that cause genetic disease
Gene therapy for dominant alleles
- sufferer will be heterozygous
- they will have the functional allele
- silence dominant allele
- use a vector to add a DNA fragment into dominant allele
- dominant allele won’t be transcribed
- recessive allele expressed
Gene therapy for recessive alleles
- sufferer will be homozygous
- use a vector to add the functional allele to DNA
- dominant allele will be expressed
Possible problems with gene therapy
- allele insert into wrong locus
- could silence wrong gene by mistake
- Gene could be over expressed
- use of gene therapy could be used for non-medical uses
Germ line gene therapy
Change the allele of gametes
Inheritance from germ line gene therapy
- all future offspring inherit
- illegal in humans
Somatic gene therapy
Changing the alleles of body cells (non gametes)
Inheritance from somatic gene therapy
Offspring do not inherit change
Mutation
A change to the nucleotide sequence of DNA
Mutagenic agent
Increase the rate of mutations
Base analogs
A chemical that can substitute from a normal nucleotide base
Radiation
Change the structure of DNA
Change DNA bases
Chemicals that react with DNA to change its structure
Substitution
One base is swapped for another
- no change, single amino acid changed
Addition
An extra base is added
Deletion
A base is removed
- frame shift, all the following triplets in the sequence will be affected
Inversion
A sequence of bases is reverse
- no change, change a few amino acids, no frame shift
Duplication
One or more bases are repeated
Translocation
A sequence of DNA is removed from one part of the genome and moved to another part of the genome
Changes to the base sequence of DNA
- no change to amino acid sequence
- change to a single amino acid
- change to sequence of many amino acids
Why is there no change to amino acid sequence after a mutation
- amino acids are coded for by more than one triplet on DNA
- it is degenerate
Why is there only change to a single amino acid after a mutation
- changes to one DNA triplet
- changes the translation of one amino acid
- changes primary structure of the protein
- change the hydrogen/ ionic bonding
- change the tertiary structure of the protein
Why is there a change to the sequence of many amino acids
- frame shift changes the sequence of all the following DNA triplets
- change all the following amino acids in sequence
- changes the primary structure
- changes the hydrogen/ ionic bonding
- change tertiary structure
Stem cells
Can divide and differentiate to become different types of cell
Totipotet
Stem cells that can differentiate into any type of specialised cell
Where are totipotent cells from
- early mammalian embryos
- can form placental cells
Pluripotent
Stem cells that can differentiate into many types of specialised
Where are pluripotent stem cells from
- embryos
- can’t make placental cells
Multipotent
Stem cells that can differentiate into a few types of specialised cells
Example of multipoint cells
bone marrow to RBC and WBC
Unipotent
Stem cells that can differentiate into one type of specialised cell
Example of unipotent cells
Heart unipotent cells can make cardiomycetes only
Cell specialisation/ differentiation
- all cells contain 100% of an organisms DNA
- conditions within cells control which genes are expressed into proteins
- change internal environment of the cell - affect the expression of other genes (are specialised)
iPs cells
Treating unipotent stem cells with transcription factors that make them become pluripotent
How to make induced pluripotent stem cells
- use a modified virus as a vector
- virus inserts transcription factor genes from pluripotent cells into the DNA of unipotent stem cells
- transcription factors are expressed
- ultimate goal is to make totipotent stem cells this way
Creation of embryonic stem cells
- embryos are made in a lab by IVF
- pluripotent stem cells are removed after a few days
- embryo is destroyed
- pluripotent stem cells can differentiate into all types of body cells
Creation of adult stem cells
- taken from adult in an operation
- adult stem cells are multipoptent
- less useful for medicine as they can’t form every type of cells
Why are stem cells used
They can differentiate to form any type of specialised cell
Bone marrow transplants
- contains multipotent stem cells
- can differentiate into RBCs and WBCs
- if someone has fault bone marrow you can replace there bone marrow to form new healthy cells
Growing new organs with stem cells
- no donors needed
- iPs cells
- iPs organs have the same antibodies so there is no organ rejection
Advantages of stem cells in medicine
- saves lives
- improve quality of life
- prevent suffering
Ethical issues with stem cells in medicine
- they are taken from IVF embryos which could be developed into a foetus if implanted
- some people believe from fertilisation a zygote has a right to life
- adult stem cells are not pluripotent
- iPs cells could be the future solution
Transcription factors
Proteins that control the rate of protein synthesis by switching some genes on and other genes off
Promoter region
Short sequence of DNA at the start of a gene
Stages of activators and repressors
1) transcription factor moves from the cytoplasm into the nucleus
2) binds to the promoter region
3) activators - help RNA polymerase to bind to DNA ( gene is transcribed)
4) repressors - prevent RNA polymerase binding to DNA (gene is not transcribed)
Controlling transcription factors - oestrogen
- oestrogen is a steroid hormone
- oestrogen binds to a receptor called an oestrogen receptor
- forms oestrogen-oestrogen receptor complex
- transcription factors can also be turned on/off by second messengers
Cancer
Uncontrolled cell division
How do tumour suppressor genes work
- proteins that slow down the rate of mitosis or speed up the rate of apoptosis
- if theres a mutation into the tumour suppressor gene
- the protein might be non-functional
How to proto-oncogenes work
- make proteins that increase the rate of mitosis
- if a proto-oncogene mutates it is called an oncogene
- oncogenes can be over expressed
Epigenetics
Changes to gene expression caused by environmental factors
How does epigenetics work
- controls gene expression by preventing transcription
- can be inherited between generations
- helps organisms respond to changes in their environment
Methylation of DNA
More Methylation = Terminate Transcription
- methyl can attach to DNA at CPG sites
- Methylate CpG sites prevent transcription enzymes attaching
- transcription is prevented
Affects of acetylation of histones
Less acetylation of histones Terminates Transcription
How does the acetylation of histones work
- acetyl groups make histones space out
- allows transcriptional enzymes to attach - gene is expressed
- enzymes remove acetyl groups - prevents transcription
What does RNAi do
Controls gene expression by preventing translation
siRNA - small interfering RNA
- short double stranded RA
- combines with proteins to form a siRNA-protein complex
- siRNA is single stranded
- siRNA has complementary base sequence to target mRNA
- siRNA protein complex breaks down the mRNA into pieces
- prevents translation
- mRNA pieces are recycled
MicroRNA
- microRNA combines with a protein to form a microRNA-protein complex
- bind to mRNA by complementary base pairing
- less specific that siRNA - works on more than one mRNA
- prevents translation by stopping the ribosome attatching
- mRNA can be stored and used later or recycled
Hypermethylation
Tumour suppressors genes
- means that proteins not transcribed
Hypomethylation
Proto-oncogenes
- more protein is transcribed
Benign tumours
- non-cancerous
- grow slowly
- harmless
- can become malignant
Malignant tumours
- are cancerous
- grow quickly
- destroy tissue
- can break up and spread in blood/ lymphatic system
Oestrogen and breast cancer
- high levels of oestrogen can cause some types of breast cancer
- oestrogen can bind to proteins to form a transcription factor called oestrogen-oestrogen receptor complex
- increase rate of cell division, increase DNA replication, increase mutation, increase cancer
Identifying tumour cells
- mitosis - more cells dividing
- nuclei - large and odd shaped, more than 1 per cell
- irregular cell shape
- loss of normal function
- disorganised arrangement
Genome
The complete set of genetic material that an organism has
Proteome
The complete set of protein that an organism can make
Sequencing projects
- human genome project - sequenced whole human genome
- only sequence short fragments at once
- split genome up into small sections -> sequence -> put back together
- sequencing methods are constantly being up dated
Predicted amino acid sequence - simple organisms
- few regulatory genes
- little non-coding DNA
Predicting amino acid sequence - complex organisms
- regulatory gene turns on/off other genes
- lots of non-coding DNA
- hard to determine proteins from DNA
Uses of genome projects
- understanding evolutionary relatedness
- medicine - understand the antigens to develop new vaccine
uses of genetically modified organisms in agriculture
- express protein from bacteria
- protein is toxic to insects
- fewer insects eat soya plants
- gene from corn to rice expresses vitamin A
advantages of using genetically modified organisms in argiculture
- uses less chemical pesticides
- more efficient food chain
- prevents blindness caused by vitamin A deficiency (corn to rice gene)
disadvantages of using genetically modified organisms in agriculture
- monocultures have a low genetic diversity
- susceptible to environmental factors eg disease
- have to buy seeds every year
- decrease in biodiversity
uses of genetically modified organisms in industry and research
- making enzymes eg lipase
- transformed pathogen to TREAT disease - attach other pathogens but don’t infect humans
advantages of using genetically modified organisms in industry and research
- reduce energy and cost
- fast and cheap production
- treat disease
- pathogens won’t develop resistance
- reduce suffering
disadvantages of using genetically modified organisms in industry and research
- could mutate and infect humans
- could be used in a negative way
uses of genetically modified organisms in medicine
- transform bacteria to express proteins
- mammals can also be transformed to produce useful products in their milk
advantaged of genetically modified organisms in medicine
- makes human proteins
- cheaper and easier than making protein synthetically
disadvantages of using genetically modified organisms in medicine
- possible unexpected problems
- using animals as commodities
DNA probes
a short sequence of DNA that is complementary to a specific allele/ mutation / gene
uses of DNA probes
to test if a sample of DNA contains a specific sequence
stages of using a DNA probe
1) attach a label to DNA probe
2) is sequence is present than DNA probe will hybridise and stick to DNA sample with a complementary sequence
3) rise to remove unhybridized DNA probes
- DNA must be single stranded
DNA microarray stages
1) many DNA probes attached to a tile in a grid
2) add patients DNA with label
3) DNA will hybridise to complementary DNA
- 4) rinse and view labels
How do you use DNA microarry
- attach the label to human DNA
- test multiple alleles at once
DNA probes in genetic counselling
- used to identify: carriers of a genetic disease, specific alleles, the most effective treatment
- healthcare professionals can advise people about the risks of passing on inheritable diseases, developing diseases later on in life, making informed decisions about treatment and preventions
uses of DNA probes in genetic screening
- parents can see if they are carriers of recessive alleles
- you can diagnose and treat before symptoms show
uses of DNA probes in personalised medicine
- if a doctor knows a patients genotype it gives the best drugs for them
- change drugs if you know a patient has an allele that causes a side effect with the normal drug
genetic fingerprinting
identifying individuals by comparing the differences in their Variable Number Tandem Repeats (VNTR)
genetic fingerprinting in forensics
- sample of DNA from a crime scene and suspects
- amplify using PCR
- run gel electrophoresis
- the chances of two individuals having the same number of VNTR’s is very low
- suspects will have more matches to the crime scene
genetic fingerprinting for relatedness
- the more closely related two individuals are the higher the percentage match of VNTRs
- conservation of endangered species to avoid inbreeding by mating individuals who are least related to help maintain genetic diversity
genetic fingerprinting for medical diagnosis
- genetic fingerprints can be used to test for specific combinations of alleles
- can be used to diagnose genetic disorders
facts of genetic fingerprinting
- non-coding DNA contains lost of VNTRs
- don’t code for proteins
- don’t affect phenotype - vary more than coding DNA
- the number of repeats of each VNTR varies
