Topic 7: Modern Genetics Flashcards
Define genome
The total of all the genetic material in an organism
Where is the DNA found in prokaryotes?
In plasmids/chromosomes
Where is the DNA in eukaryotes?
In the nucleus, mitochondria and chloroplasts
What are exons?
Coding regions of DNA
What are introns?
Non-coding regions of DNA
Define gene sequencing
A method of analysing the individual base sequence along a DNA strand or an individual gene
What does PCR stand for?
Polymerase chain reaction
What is the purpose of PCR?
To amplify small samples of DNA by making more copies of it. Makes it easier to analyse
Summarise the stages of PCR
- Add DNA sample to mixture of enzymes, primers + nucleotides
- Heat mixture to 94 degrees
- Cool mixture to 55 degrees
- Raise temperature to 72 degrees
Why is taq polymerase used in PCR rather than normal DNA polymerase?
Thermicus aquaticus is an extremophile which is adapted to being at high temperatures. Its polymerase doesn’t denature at the temps of PCR like normal DNA poly.
What is Taq an abbreviation for?
Thermic aquaticus (a bacteria)
What are primers?
Small sequences of DNA that must join to the beginning of the strand before copying can begin. Act as a starting point for DNA synthesis.
What is needed in the mixture that DNA is added to before PCR begins?
Taq polymerase
Primers
Buffer
Good supply of four bases
Why is the DNA initially heated to 94 degrees in PCR?
To break the hydrogen bonds between the DNA strands so that they become available for replication
Why is the temperature cooled to 55 degrees in PCR?
Allows primers to bind to complementary regions on the DNA
Why is the temperature raised for a second time to 72 degrees in PCR?
Optimum temperature for the Taq polymerase. Allows it to add onto the primers and extend the DNA strands
What are the uses of PCR?
DNA profiling
Gene sequencing
What is gene sequencing?
A method to determine the base sequence in a particular gene
Explain the basic principles in gene sequencing
- DNA cut up into fragments
- PCR done in mixture of normal and terminator bases
- Terminator bases have fluorescent dye to mark them
- Different length fragments made
- Separate with gel electrophoresis
- Compare to a DNA ladder to find nucleotide positions
What are the uses of DNA sequencing?
Predicting the amino acid sequence of a protein
Understanding genes and proteins used in non-communicable diseases
What is DNA profiling?
A method by which people can be identified and compared via the patterns in their DNA
What are satellites?
Short sequences of DNA that can be repeated up to 100s of times in the introns
What are micro-satellites?
Short sequences of DNA between 2-6 bases long that can be repeated between 5 and 100 times in the introns
What are mini-satellites?
Short sequences of DNA between 10-100 bases long and repeated 50-100s times in the introns
What principle is DNA profiling based on?
While everyone has satellites on the same loci on their homologous chromosomes, the number of times they are repeated differs between everyone
Summarise the stages of DNA profiling
Isolate DNA Restriction enzymes Gel electrophoresis Southern blotting Gene probes
What are restriction endonucleases?
Special enzymes that cut DNA at recognition sites on the intron sequence
Why are restriction endonucleases added to the DNA being profiled?
To cut around the repeating satellites and make different sized fragments which can then be separated out by gel electrophoresis
Summarise the stages of gel electrophoresis
DNA samples put into wells in agarose jelly with dye
Known DNA samples put in for comparison
Current applied
-vely charged DNA attracted to the +ve anode
Bands separate
Shone under UV light and identified
Why is DNA negatively charged?
Bc of the negatively charged phosphate group
Why is dye added to the DNA in gel electrophoresis?
So that the DNA can be seen on the plate
What is the relationship between size and speed of movement of DNA fragments in gel electrophoresis
Smaller fragments move faster and further
Explain the process of Southern blotting
Alkaline solution denatures DNA and separates it
Nylon filter put on it and draws up DNA - appears as blots
DNA covalently bound to filter using UV light
What are gene probes?
Complementary pieces of DNA which are labelled with a fluorescent molecule or radioactive isotope
What information can be found from the DNA profile produced in DNA profiling?
The number of microsatellites per fragment and the number of repeats
What are the uses of DNA profiling?
Paternity testing
Forensic science
What are housekeeping proteins?
Proteins found in all cells regardless of differentation
In what ways can you study cell differentiation?
Gel electrophoresis
Gene probes
How can you use gene probes to find a specific gene?
Specific probe is used for a specific gene Heat DNA Mix with fluorescently labelled mRNA Hybridise at complementary points Probe is labelled so it marks the genes
What are the two stages involved in the expression of a gene
Transcription
Translation
What are transcription factors?
Proteins that bind to DNA in the nucleus at promoter regions and control transcription. Can turn a gene on or off
What are promoter regions?
Areas on DNA which transcription factors bind to. Usually found right before the starting point of the gene
What are repressors?
Transcription factors which block transcription
How do enhancer sequences work?
Enhancer sequences change the structure of the chromatin of DNA which makes it easier or harder for RNA polymerase to bind
What is RNA splicing?
Post-transcriptional modification of mRNA
What happens in RNA splicing?
Introns are removed (and sometimes exons) and exons are put together
What are spliceosomes and what do they do?
They’re enzymes which join exons together to form functional mRNA
How can one gene make many different proteins?
Because the exons of the gene can be joined together by the spliceosome in different ways. Means a different order of the bases so different amino acid sequence so different protein
What is epigenetics?
The study of changes in organisms due to modifications of gene expression rather than alteration of the genetic code
Name some intracellular systems which interact to control genes
DNA methylation
Histone modification
Non-coding mRNA
Name all the ways in which gene expression is controlled
RNA splicing Transcription factors DNA methylation Non-coding mRNA Histone modification
What is DNA methylation?
The addition of a methyl group to a DNA molecule
Where are methyl groups added on DNA?
Only on cytosine
What enzyme is needed in DNA methylation?
DNA methyltransferase
How does DNA methylation affect genes?
It silences the genes so they become inactive because it changes the shape of the nucleotides so transcription factors and RNA polymerase can’t bind
What is DNA demethylation do?
Removes a methyl group from DNA and activates the gene
How can DNA methylation lead to disease?
Can suppress important genes such as tumour suppressor genes which leads to cancerous tumours growing.
What is histone acetylation?
Addition of an acetyl group to histone
What does histone acetylation produce?
Euchromatin - regions of chromatin with high transcriptional acitivity bc structure is loose and the chromatin is activated.
What does histone methylation produce?
Heterochromatin where chromatin is tightly coiled so regions have low or no transcription
What is non-coding RNA?
RNA transcribed from the non-coding regions of the chromosomes
How can ncRNA control transcription?
Coats chromosomes and deactivates it because of supercoiling which prevents transcription
What are stem cells?
Undifferentiated cells with the potential to develop into many different types of specialised cells from instructions in their DNA
What does totipotent mean?
Cells that are capable of producing every kind of adult and placental cell in the embryo
Give an example of totipotent cells
Embryonic stem cells
What does pluripotent mean?
Capable of producing almost every type of cell
Give an example of pluripotent cells
Stem cells found in the umbilical cord
What are multipotent cells?
Cells which can form a limited number of actdult cells
Where can adult stem cells be found?
In the tissues and organs of adults
What are the problems with adult stem cells?
Multipotent
Found in small cells
Hard to extract
What are the advantages of embryonic stem cells over other stem cells?
Totipotent so they can form a wide range of cells
Stay totipotent after cell division
What does the type of tissue a stem cell forms depend on?
The location of the stem cell
Describe the develop of an embryo to a somatic stem cell including what type of stem cell is formed
Embryonic stem cell (totipotent)
Blastocyst stem cell (pluripotent)
Fully differentiated somatic stem cell (multipotent)
What controls the changes that occur to stem cells?
Gene expression (controlled by epigenetic factors)
Give an example of epigenetic control in humans?
Turning fetal haemoglobin to adult haemoglobin by turning of the fetal globin strands and turning on the adult ones
Name the uses of stem cells in medicine
Stem cell therapy: new body parts and repairing damage to current body parts
Therapeutic cloning
Why are adult stem cells used in stem cell therapy over embryonic stem cells?
Easier to control the differentiation and is less likely to lead to cancer
How are stem cells used to make new body parts?
Grown on either a synthetic or collagen based framework and forms body part
What is the advantage of using stem cells to make new body parts?
Because they’re the body’s own cells they won’t be rejected
What is therapeutic cloning?
An experimental method of producing large quantities of stem cells which can be used to treat diseases caused by fault genes.
Describe the method of therapeutic cloning
Remove body cell nucleus
Put into an ovum which has had body cell removed
Apply electric shock
Ovum divides and develops into embryonic stem cells
SCs cultured into required cell
Genetic diseases removed and cells inserted back into the patients
Why is an electric shock applied to the ovum in therapeutic cloning?
To fuse the nucleus and the ovum
To trigger the development of the ovum
What are the advantages of using stem cells in therapeutic cloning
Patient’s own cells so they won’t be rejected
Don’t need to wait for a donor
Diseases can’t be treated that couldn’t before
What are the disadvantages of using stem cells in therapeutic cloning?
Experimental
Can cause cancer
Embryos destroyed in the process - unethical
What does iPSC stand for?
Induced pluripotent stem cells
How are iPSCs made?
Fibroblast cells had four genes coding for transcription factors put into them by a harmless retrovirus vector. Genetically engineered from multipotent to pluripotent
What are fibroblast cells?
Cells that are relatively unspecialised
What are the advantages of iPSCs?
No ethical objections bc no embryo is damaged
Not rejected by body
Source of pluripotent stem cells which can give rise to many cells
Divide indefinitely
What are the disadvantages of iPSCs?
Experimental
Low success rate
Expensive
Still a chance of becoming cancerous
What is genetic engineering?
Changing the genetic material of an organism by inserting genes of a different organism into it
What is recombinant DNA?
DNA which has been artificially made by combining constituents from different organisms
Describe the stages of producing recombinant DNA
- isolate the gene
- insert it into the vector DNA
- ligase glues it together
- introduced to host cell
How can genes be isolated when producing recombinant DNA?
Reverse transcriptase
Restriction endonucleases
Explain how reverse transcriptase can be used to isolate genes
Reverses the transcription of mRNA to produce an artificial strand of cDNA
Explain how restriction endonucleases can be used to isolate genes
They cut DNA at specific sites, making small pieces of DNA which are easier to work with
Form sticky ends which can be used to bind to the vector DNA
What are sticky ends and why are they needed?
Overhangs of exposed bases which are needed to form hydrogen bonds to complementary sticky ends on the vector DNA
Why is the vector DNA cut with the same restriction endonucleases when producing recombinant DNA?
So that it has complementary sticky ends to the sample DNA
What does DNA ligase do?
Sticks the sample DNA and the vector DNA together by reforming the sugar phosphate backbone
In what ways can you identify recombinant bacterium?
Using fluorescent markers which fluoresce under UV light
Replica plating
Explain the process of replica plating
Recombinant DNA is inserted into the plasmid which also carries genes for antibiotic resistance
Bacteria grown on master plate in complete media
Inverted and imprinted onto smooth velvet surface
Imprinted onto a fresh colony
Grown in different media
Compare to the master plate
What is the function of a vector?
To artificially carry foreign genetic material into another cell where it can be transcripted and translated
What are the characteristics of a good vector?
Targets correct host cell
No adverse side effects
Ensures the DNA is transcribed
Name vectors that can be used to produce recombinant DNA
Gene guns Viruses Liposome coating Microinjection Agrobacterium
Explain how gene guns work
The DNA is coated onto a gold/tungsten pellet which is then shot at high speed into the cell
Explain how viruses act as vectors
Harmless virus is engineered to carry a desirable gene which is then inserted into animal cells.
Explain how liposome coating acts as a vector
Gene is coated in liposome so it can fuse and pass through the phospholipid bilayer and pass on DNA
What are liposomes?
Spheres formed from the lipid bilayer
How can microinjection act as a vector?
DNA can be injected into cells using a micropipette then manipulated using a micromanipulator
What happens in knockout organisms?
A gene is silenced and the function that is then lost is observed
Why do we use knockout organisms?
To identify the function of a gene (through trial and error) and to use them as models for disease bc we can test on them for cures
Explain how mice are used as knockout organisms
Adult mice have embryonic stem cells
Modified so they’re heterozygous for the gene
Bred to form homologous offspring who have lost that function
How do you know which of the knockout mice are carrying the homozygous gene?
The silenced gene has a marker on it
Which diseases can we use knockout mice to study?
Cystic fibrosis
Cancer
Parkinson’s
Obesity
Generally, why have soya beans been genetically modified?
To improve the quality and yield of the crops
Have herbicide resistance
Have more oleic acid and less linoeic acid
Describe the method by which soya beans are genetically modified
Ti plasmid extracted from bacterium
Target gene inserted into plasmid
Inserted back into bacterium
Plant infected. Tumour grows with cells with new gene
Sample of cells taken and cultured to make whole new plants
What bacteria is used in the genetic modification of soya beans
Agrobacterium tumefaciens
What does the bacterium do in the genetic modification of soya beans?
Causes tumours called crown galls
Why are fatty acids altered when genetically modifying soya beans?
To stop oxidation of soya beans
Why do you want more oleic acid in soya beans?
Because they aren’t easily oxidised
Monounsaturated
Why do you want less linoleic acid in soya beans?
Easily oxidised
Polyunsaturated
What are the main concerns with genetically modifying foods such as soya beans?
- ingesting foreign DNA
- unethical bc somtimes animals are used
- GM makes seeds infertile so new ones have to be used
- build up of antibiotic resistance
- new genes spread uncontrollably
What are terminator bases?
Dideoxynucleotides - hydroxyl group missing from carbon three so they stop DNA synthesis
Similarities between iPS cells and embryonic stem cells?
Give rise to many different type of cells
Divide indefinitely
Differences between iPS cells and embryonic stem cells?
iPS - adult, embryonic - younger
iPS - pluripotent, embryonic - totipotent
iPS - no rejection, embryonic - rejection
iPS from adult cells but embryonic cells from inner mass of morula
How do vectors carrying recombinant DNA get taken up by cells?
Mix bacterium and vector
Applying heat treatment
How are DNA profiles compared?
Looking at the total number of bands
Band width
Position of bands
