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How is DNA replicated on a large scale
PCR
What does PCR stand for
Polymerisation chain reaction
In PCR what’s the first step and why
DNA is heated to 96 degrees to denature the strands
What happens in the second stage of PCR and why
The reaction is cooled to 60 degrees allowing primers to anneal to the DNA strands
What happens in the final stage of PCR and why
The reaction is heated to 72 degrees allowing the nucleotide chain to be extended using DNA polymerase
How many times is PCR usually repeated
25-35 times
What’s gel electrophoresis
A technique used to separate DNA fragments by size
What are introns
Large non-coding regions of DNA
What are exons
The DNA that codes for proteins
What are VNTR’s
Sequences of DNA 20-50 base pairs long that are repeated 50-100 times
What are VNTR’s also known as
Minisatelites
What are STR’s
Sections of DNA 2-4 base pairs long repeated 5-15 times
What are STR’s also known as
Microsatelites
Why are exons not often used in genetic profiling
Because the nucleotide differences are too small
What are the 5 key components in PCR
DNA polymerase, primers, free nucleotides and a DNA sample
What’s different about taq DNA Polymerase
It can withstand the temperatures used in PCR due to its thermal stability
How does gel electrophoresis work
DNA is negatively charged due to its phosphate group and so moves along the agarose gel towards the positively charged anode, different DNA fragments have a different number of repeats and so travel different distances along the gel
What are 3 key differences between gel electrophoresis and TLC
TLC separates by solubility rather than size
TLC separates non-charged particles
Electrophoresis requires an electric current TLC doesn’t
Why do smaller fragments travel further through the agarose gel
They can fit through the pores in the agarose gel more easily than larger fragments
Where is the CFTR protein located
The integral membrane protein
What’s the difference between a normal CFTR and mutant CFTR channel
In a mutant CFTR channel chloride ions aren’t transported outside the cell, causing the mucus to become thicker and stickier due to a lower water content
What are some uses of DNA profiling
In forensic science, paternity tests and species identification
Who was Frederick Sanger
The first scientists sequence nucleic acids from viruses and bacteria
What did Sangers discovery lead to
Capillary tube electrophoresis
What allowed the human genome project to be completed ahead of the initial timeframe
The introduction of new faster and more powerful computers
What’s DNA sequencing
The technique of determining the precise corded of nucleotides within a DNA molecule
What are terminator bases
Nucleotides tagged with a fluorescent marker that stop DNA polymerase working
What are the 5 steps involved in genome sequencing
The DNA is mixed with a primer, DNA polymerase, an excess of free nucleotides and terminator bases
The mixtures is put into a thermal cycler
Free nucleotides are added to the DNA strands but when a nucleotide with a terminator base is added the DNA can’t be extended any further
Many copies of different lengths of DNA are produced with different coloured tags at the end
The DNA fragments are separated by gel electrophoresis and a laser is used to record the tag colour
How does Next-generation sequencing work
Sequencing occurs on a flow cell instead of in a capillary tube allowing millions of DNA fragments to be replicated in situ using PCR forming clusters of identical DNA fragments
What’s bioinformatics
the development of the software and computing tools needed to organise and analyse raw biological data
How can bioinformatics be used in relation to an organisms genome
Using a high-powered computer a database can be formed sequencing an organisms genome allowing comparisons between organisms
What’s computational biology
Using the data produced by bioinformatics to build theoretical models of biological systems
How is computational biology used in relation to an organisms genome
It can be used in the analysis of the data from sequencing the billions of base pairs in DNA for working out the 3D structure of proteins
What’s genomics
The field of genetics that applies DNA sequencing methods and computational biology to analyse the structure and function of genomes
What are some applications of analysing the genomes of pathogens
Allowing doctors to identify the source of an infection or identify antibiotic-resistant bacteria and allowing scientists to identify regions of the genome that may be useful in the development of new drugs
What’s DNA barcoding
Identifying particular sections of the genome that are common to all species but vary between them
What region of genome is used for DNA barcoding in animals
A section of the mitochondrial DNA in the gene cytochrome c oxidase
What organisms is DNA barcoding not suitable for and why
Fungi and bacteria as there have not been suitable regions identified
how can scientists calculate how long ago two species diverged from a common ancestor
By calculating the basic mutation rate of DNA from two different organisms
What’s proteomics
The study and amino acid sequencing of an organisms entire protein complement
How do scientists know that genes can code for multiple different proteins
Because there are a smaller number of coding genes in the human genome than proteins
What are spliceosomes and what do they do
They’re enzyme complexes that join together exons to form mature functional mRNA
How do spliceosomes contribute to different phenotypes
They can join the same exons in a variety of different ways producing different versions of functional mRNA
How can protein modification help explain the relationship between genotype and phenotype
Proteins can be modified by other proteins after being synthesised allowing them to be lengthened or shortened and even form different proteins
what’s synthetic biology
the design and construction of novel artificial biological pathways, organisms or devices or the redesign of existing natural biological systems
What different techniques does synthetic biology include
Genetic engineering, using biological systems in industrial contexts, synthesising new genes to replace faulty ones and synthesising new organisms
What’s genetic engineering
when the genes from one organisms are transferred into another organisms using a vector in such a way that the genes are expressed
What are the key steps in the genetic engineering process
Identifying the desired gene, isolating the plasmid, isolating the gene of interest (cutting this and the plasmid using the same restriction endonuclease), inserting the gene into the plasmid using DNA ligand, insert the plasmid into bacterium and reproducing the GMO’s
What’s a transgenic organism
An organism with nucleotide sequences from a different species
What are restriction endonucleases used for
To cut genes at specific base sequences
What’s the function of DNA ligase
To join together the cut ends of DNA by forming phosphodiester bonds
What’s the function of reverse transcriptase
To build double stranded DNA from single stranded RNA
What’s a vector
Something used to deliver DNA fragments into a cell
What are 3 common vectors
Plasmids, viruses and liposomes
How is vector success monitored
Using marker genes
What are 2 common marker genes
Antibiotic resistance or fluorescence
What’s electroporation
Using small electric currents to make bacterial membranes permeable allowing plasmids to enter the cell
Why must the electric current be controlled during electroporation
To avoid permanent damage to the cell membrane
What are the 3 main methods of producing DNA fragments
Direct synthesis of DNA from a proteins primary structure
Converting mRNA to cDNA
Isolating the gene from its DNA sequence
What enzyme is used to form cDNA
Reverse transcriptase
What type of ends form when restriction endonucleases cut at the same point on both sugar-phosphate backbones
Blunt ends
What type of ends form when restriction endonucleases cut one sugar-phosphate backbone longer than the other
Sticky ends
Why do sticky ends bond together more easily than blunt ends
Because hydrogen bonds can form between the exposed bases on the identical sticky ends
What’s ligation
Sticking 2 strands of DNA together
What method is used to identify whether bacterial plasmids contain the desired gene
Replica plating
how are marker genes used to indicate whether a DNA fragment has been successfully inserted
The DNA fragment is inserted into a marker gene and if this is done successfully the marker gene won’t function
What’s electrofusion
Tiny electric current are applied to the membranes of two different cells to fuse the cell and nuclear membranes of the two cells
What must be inserted along with a marker gene
Promoters to ensure that the gene is transcribed
Why is it easiest to access bacterial DNA
There are no histones so the DNA is easily accessible
What are some limitations of using bacterial DNA in genetic engineering
There are no introns and therefore no mRNA splicing
What’s an example of genetic modification in plants
Golden rice wheee beta carotene was inserted into rice
What are the ethical concerns associated with genetic modification of microorganisms
GM microorganisms could be used in biological warfare
What are the benefits of GM microorganisms
The production of medicines, antibiotics and increasing the food supply for the growing population
Explain how soya beans have been genetically modified
A gene has been inserted into soya beans that produces the Bt protein which is toxic to many pests that attack the plant increasing crop yield
How has patenting affected the usefulness of GM crops
Many crops including GM soya beans have been patented making them more expensive and less accessible to people in less economically developed countries the seeds must also be re bought every year and cannot be replanted
What are micro injections and how are they used in GM
DNA covered in gold can be injected into organisms introducing new genes to animal DNA
What are examples of GM animals
Swine fever-resistant pigs and faster-growing salmon
What’s pharming
the production of human medicines in animals
What are the 2 main ways pharming is used
Creating animal models
Creating human proteins
What are ethical issues associated with pharming
Is it right to put human genes into animals? Is welfare compromised during the production of genetically modified animals?
What’s somatic gene therapy
Replacing a mutant allele with a healthy allele in the affected somatic cells
What’s a limitation of somatic cell gene therapy
It’s only a temporary treatment because somatic cells have a limited life and are replaced from stem cells with the faulty allele
What’s germ line cell therapy
treating a disease by inserting a healthy allele into the germ cell
Why is germ line cell therapy not practised
Due to the ethical and medical concerns around intervening with germ cells
What’s gene therapy
Fixing cellular problems by replacing faulty genes in somatic or germ line cells by inserting a normal one
What’s a germ cell
A pre-gamete cell involved in reproduction
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