Topic 7 Flashcards
Define genome
Genome: the total of all the genetic material in an orga
What is PCR and how does it occur?
Polymerase Chain Reaction (PCR): the reaction used to amplify a sample of DNA, to make more copies of it very rapidly.
- Denaturation: DNA sample is heated to 95 degrees to break hydrogen bonds and separate it into two strands.
- Annealing: DNA sample is cooled to 54 degrees, allowing primers to attach to opposite ends of the target sequence. (Primers are small sequences of DNA that must join to the beginning of the separated DNA strands before copying can begin. They are added because a DNA molecule is antiparallel and Taq only works in one direction)
- Elongation: DNA Taq copies the strands, when the mixture is heated to 72 degrees. (This is the optimum temperature for the Taq DNA polymerase enzyme to build the complementary strands of DNA)
- One cycle of PCR yields two identical copies of the DNA sequence. Therefore the process is repeated 30 times to yield 2 30 copies of DNA. That is over 1 billion copies of DNA.
PCR gives amplified DNA samples and is used because it enable scientists to carry out analysis of DNA, without fear of using it up.
What is DNA sequencing and how does it occur?
DNA Sanger Sequencing: reveals the order of the bases in a segment of DNA
- A mixture containing single stranded DNA, DNA polymerase, the four deoxyribonucleotides (A T G and C ),and a single short primer is prepared.
- Equal amounts of this mixture is placed in four tubes. A different dideoxyribonucleotide is added to each tube.
- When a deoxyribonucleotide is added into the chain, replication continues. When a dideoxyribonucleotide is inserted, strand synthesis is terminated.
- The contents of the reaction tubes are then transferred to layer of an electrophoresis gel and oligonucleotides are separated by size and nucleotide type.
- The shortest oligonucleotide moves furthest down the gel. Reading from bottom to top one base at a time, prices the correct DNA sequence.
What is DNA profiling and how does it occur?
DNA Profiling: the identification of repeating patterns in the non-coding regions (introns) of DNA.
- PCR to amplify DNA sample.
- Strands of DNA are cut into fragments using restriction endonuclease. These enzymes cut the DNA at particular points in the intron sequences.
- This gives a mixture of different sized DNA fragments.
- These fragments are separated and identified using Gel Electrophoresis.
- The DNA fragments are mixed with a dye and placed in wells in agarose gel, with known DNA fragments to aid identification.
- An electric current is passed though and the DNA fragments move towards the positive anode, at different rates according to size. Smallest fragments move furthest.
- Southern blotting then takes place.
- DNA fragments are drawn from the gel to a filter, leaving the DNA fragments as ‘blots’ on the filter.
- A DNA profile is produced as a graph with each peak representing the number of microsatellites in a fragment.
What are introns and exons?
Introns are repetitive noncoding regions between the genes. Within introns are short sequences of DNA that are repeated many times to form micro and mini satellites. The same micro and mini satellites appear in the same positions (loci) on each pair of homologous chromosomes. The number of repeats vary; inherited from parents, two individuals never have the same pattern of DNA (unless identical twins).
Introns are used instead of coding exons, because introns have much greater variation. I.e exons code for the same proteins within the same species.
How can DNA profiling be used to identify criminals and in paternity testing?
To identify criminals: A DNA profile is created using evidence left at the crime scene. E.g blood left behind. The profile is then compared to the suspects profiles.
In paternity testing: A DNA profile is taken from the mother, suspected father and child. The child must have all its micro satellites from with its mother or father. Hence if some do not match , then the suspect is no the father.
What are transcription factors? How do they control gene expression?
Transcription factors: proteins that bind to the DNA in the nucleus and affect the process of transcribing the genetic material.
The most common way of controlling gene expression is by switching on and off transcription of certain genes.
Transcription factors have DNA binding regions that enable them to bind to specific regions on the DNA known as promoter sequences.
Some transcription factors stimulate the transcription of a region of DNA simply by binding to a DNA promoter sequence, stimulation the start of transcription of that area of the DNA.
Other transcription factors bind to enhancer sequences and regulate the activity of the DNA by changing the structure of the chromatin, making it more or less open to RNA polymerase.
What is RNA Splicing. How does it control gene expression?
Pre mRNA is the mRNA that is transcribed directly from the DNA before it has been modified. It still contains introns.
Hence RNA splicing takes place. It is the process by which introns are removed from the mRNA made during transcription.
Spliceosomes are enzyme complexes that join together the exons to produce mature functional mRNA.
the ‘looped’ intron is then removed.
Spliceosomes may join together exons in many different ways. Hence, a single gene may produce several different versions of functional mRNA. These different versions of mRNA produce different proteins , hence a single gene can produce several different phenotypes.
What is epigenetics?
This refers to heritable changes in gene expression that does not involve changes to the underlying DNA sequence; it is a change in the phenotype without a change in the genotype. Epigenetics is important in ensuring cell differentiation.
Three intracellular systems that can interact to control genes include:
DNA methylation: the addition of a methyl group to a cytosine in the DNA molecule next to a guanine in the DNA chain and prevents the transcription of a gene.
Histone modification: histones can be modified in a number of different ways to affect the transcription of DNA.
Histone acetylation: the addition of an acetyl group to one of the lysine in the histone structure, which opens up the structure and activates the chromatin, allowing genes in that area to be transcribed.
Histone methylation: the addition of a methyl group to a lysine in the histone. Depending on the position of the lysine, methylation may cause inactivation or activation the the region of DNA.
Non coding RNA: 90% of the human genome is transcribed into mRNA but only 2% of those actually code for proteins. The non coding RNA seems to affect the transcription of the DNA code or modifies the product of transcription.
What are stem cells
Stem cells are undifferentiated cells that have the ability to self-renew for a continued and prolonged period of time.
What are the types fo cells?
Totipotent: an undifferentiated cell that can form any one of the different cell types needed for an entire new organism
Pluripotent: a undifferentiated cell that can form most of the cell types needed for an entire new organism.
Multipoint: a cell that can form a very limited range of differentiated cells within a mature organism.
What type of cells are in a zygote?
A zygote formed after fertilisation has the ability to become any one of the cell types in a human (totipotent). After a few sets of cell division the early embryo forms a blastocyst. The inner cell material will go on to form a new human, but it can not form the placental cells and as such is now pluripotent.
What are the uses and ethical considerations for stem cells?
Pluripotent stem cells can be found in embryos.
They provide opportunities for medical advances. For example scientists hoped they would be able to produce new tissues to fix medical issues.
BUT this has so far proved unsuccessful as it is difficult to control the differentiation of the cell. Hence the condition is solved but the patient then develops cancer.
Additionally there are ethical considerations:
immoral the embryo has the ability to grow into an adult chance should be given same human rights. not destroyed.
What are IPS cells?
IPS (induced pluripotent stem cells): adult cells that have been reprogrammed by the introduction of new genes to become pluripotent again. The genes used were; Oct4, Sox2, Klf4 and c-Myc. Hence are almost identical to embryonic stem cells.
IPS cells solve the moral issues of using stem cells and less risk of rejection. BUT the use of IPS cells is expensive as they are difficult to grow and manipulate. Risk of cancer.
How is recombinant DNA produced?
Recombinant DNA: DNA that has been formed artificially by combining constituents from different organisms.
- Restriction endonuclease is used to cut DNA strands into pieces.
- DNA ligase is used as ‘glue’ to join pieces of DNA together.
- Vectors plat a key role in the formation of recombinant DNA, transferring the required gene, into new cells.
- Plasmids are frequently used as vectors to carry the DNA into a host bacterial cell. But other vectors can be used:
- Gene guns: DNA is shot into the cell at high speed, carried on gold or tungsten pellets.
- Viruses: a harmless virus can be engineered to carry a desirable gene and then infect the animal cells, carrying DNA with it.
- Liposome wrapping: the gene to be inserted is wrapped in liposomes. Thee fue with the cell membrane and can pass through it to deliver the DNA to the cytoplasm
- Microinjection: DNA is injected into a cell through a micropipette, using a micromanipulator as a hand would be to shaky. BUT this method is still quite hit and miss.
