6.3 Flashcards
what happened in DNA research in 1969 and then in 1972
a gene was isolated from a bacterial chromsome, in 1972 a scientist sequenced a gene that codes for the protein coat of a virus. Both scientits worked from mRNA transcribed from the gene and not the raw DNA. RNA is unstable and this whole process was extrwemly slow and only worked for short genes
what happened in DNA research in 1975
the biochemist Sanger develpoed a method that ultimately allowed scientists to sequence whole genomes
what was Sanger’s approach
to use a single strand of DNA as a template for 4 experiments in separate dishes, each dish contained a solution with the 4 bases (A,T,G,C), plus an enzyme (DNA polymerase)
what was added to each dish in Sanger’s DNA sequencing approach
a modified version of one of the DNA bases was added (ddNTP), the base was modified in a way that, once incorporated into the synthesised complementary strand of DNA, no more bases could be added, each modified was was also labelled with a radioactive isotope.
whaqt happens in sanger’s DNA sequencing approach as the reaction progressed
thousands of DNA fragments of varying lengths were generated, the DNA fragments were passed trhough a gel by electrophoresis. Smaller fragments travalled further, so the fragments became shorted by length
how were the nucleotide base at the end of each fragment read in Sangers DNA sequencing
by its radioactive label. If the first one-base fragment has tymine at the end, then the first base in the sequence is T. If the two-base fragments have cytosine at the end, then the sequece is TC. If the three-base fragment ends with guanine, then the base sequence is TCG.
why did Sanger’s DNA sequencing work and what was it first used for
his method is efficient and safe. He first used it to sequence the genome of a phage virus (virus that infects bacteria cells) called Phi-X174, the first DNA based organsim to have its genome sequenced. He has to count off the bases one by one, from the bands in the gel (time consuming and costly process)
what did SAnger do in 1981, 1984 and what happened in 1995
sanger published his sequence of the human mitochondrial genome, consisting of 37 genes and 16 569 base pairs. In 1984, scientists sequenced 170 kilobase pair-long genomes of the Epstein-Barr virus. In 1995, the genome of the bacterium Haemophilus influenzae was sequenced using this approach.
In Sangers DNA sequencing how was the gene isolated (cloning DNA) and then what happened with the isolated gene
using restricted enzymes from a bacterium, the DNA was then inserted into a bacterial plasmid (the vector) and then into an Eschuerichia Coli bacterium host that, when cultured, divided many times, enabling the plasmid with the DNA insert to be copied many times.
what did each new bacterium contain when cloning DNA using Sanger’s DNA sequencing and how were these lengths of DNA isolated
a copy of the candidate gene, these lengths of DNA were isolated using plasmid preparation techniques and then they were sequenced
what happened with DNA sequencing in 1986
the first DNA sequencing machine was developed, based on Sanger’s method. Fluorescent dyes instead of radioactivity were used to label the terminal bases, these dyes glowed when scanned with a laser beam and the light signature was identified by computer. This method needed technitions to read autogradiograms.
what is high throughput sequencing and give an example
an approach to develop fast, cheap methods to sequence genomes. an example is pyrosequencing
what is pyrosequecnig and what does it involve
developed in 1996 and uses sequencing by synthesis, not by chain termination as in the Sanger method. It involves synthesising a single strand of DNA, complementary to the strand being sequenced, one base at a time, whilst detecting, by light admission, which base was added to each step.
what are steps 1-2 of pyrosequencing
- a long length of DNA to be sequenced is mechanically cut into fragments of 300-800 base pairs, using a nebuliser. 2. these lengths are then degraded into single-stranded DNA (ssDNA). These are the template DNAs and they are immoblisied.
what is step 3 of pyrosequencing
- a sequencing primer is added and the DNA is incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, apyrase and the substrates adenosine 5’ phosphosulfate (APS) and luciferin, only 1 of the 4 possible activated nucleotides, ATP, TTP, CTP and GTP is added at any one time and any light generated is detected.
what is step 4 of pyrosequencing
1 activated nucleotide (a nucleotide with 2 extra phosphoryl groups), like TTP (thymine triphosphate), is incorporated into a complementary strand of DNA using the strand to be sequenced as aa template. As this happens the 2 extra phosphoryls are released as pyrophosphate (PPi). In presence of APS, the enzyme ATP sulfurylase converts phosphate to ATP. In presence of this ATP, enzyme luciferase converts luciferin to oxyluciferin. This conversion generates visible light that can be detected by a camera. The amount of light generated is proportional to ATP availability and so indicates how many of the same type of activated nucleotide were incorporated adjacently into complementary DNA strands.
what happens to unincorporated activated nucleotides in pyrosequencing
they are degraded by apyrase and the reaction starts again with another nucleotide.
how long does pyrosequencing take
10h run generate 400 million bases of sequencing information which are assembled into longer sequences by software.
what is bioinformatics
a branch of biology that has grown out of DNA sequencing research, to store the huge amounts of data generated. Prior to computers, it was impossible to store all this information but software packages are specially designed for this purpose.
what is the human genome project
scientists predicted human genome would contain 100 000 genes. In 1990, the human genome project launched and the genome was sequenced by 2003. Scientists were surprised to learn human genome only contained 24 000 genes (not too many more than a mouse)
what dies whole genome sequencing determine
the complete DNA sequence of an organsism’s genome, in the case of eukaryote cells, that is the genetic material of the chromosomes, mitochondria and, if plants or algae, chloroplasts.
where are sequenced genomes stored
gene banks
What happened when human genome was compared to other species
it was clear that few human genes are unique to us, most of our genes were present in other organisms, we share 99% of our genes with chimpanzee, this verifies the evolutionary process
what is an examlpe of how comparism of genome has provided useful
pigs and humans have similar genes for insulin, which is why, prior to genetically-modifying bacteria to make insulin, pig insulin was used to treat patients with diabetes
what sometimes happpens as evolution progresses
some genes are co-opted to perform new tasks
what has a tiny change to a gene in humans allowed
tiny changes in human FOXP2, which is found in other mammals including mice and chimpanzees, means that humans can speak
How are differences between organisms explained at a gene level
there not different because they have completely different genes, but as some of their shared genes have been altered and no work in slightly different ways
what has altered the expression of genomes
some changes to the regulatory regions of DNA that don’t directly code for proteins, regulatory coding and genes interact in a way that, without increasing the number of genes, the number of proteins may be increased.
what has comparing genomes of thought to be closely related organisms allowed
helped confirm their evolutionary relationship or led to new info about their relationship, some cases led to organisms being reclassified.
how can animals evolutionary history be verified
DNA from bones and teeth of some extinct animals can be amplified and sequenced
what was found when extinct cave bears genome was sequenced using high throughput techniques
obtained a comparison to dogs, dogs and cave bears shared 92% of their genome
Are all humans genetically similar
yes, unless in rare cases where a gene has been lost by deletion of part of a chromosome, we all have the same genes, but different alleles
how much of out DNA is not shared with others and what does this mean
only 0.1%, although this sounds small, given that our genome contains 2 billion DNA base pairs, means there are 3 million places on DNA length where our sequences can differ due to random mutation
what is the name of the places on the DNA where these substitutions occur
single nucleotide polymorphisms, or SNPs
what is the effect of single nucleotide polymorphisms
some have no effect on the protein, some can alter a protein or alter the way a piece of RNA regulates the expression of another gene
What dies methylation of certain chemical groups in DNA play a major role in
regulating gene expression in eukaryote cells
what does mapping methylation of whole human genomes help researchers to understand
the development of certain diseases, like certain cancers and why they may not develop in genetically similar individuals. The study of this is called epigenetics
how easy is it to determine sequencde of amino acids in a protein
time consuming and laborius
how do researchers work out the primary structure of a protein
if they have the genome and know which genes code for a specific protein, by using knowledge of which base triplets code for which amino acids, they can determine primary structure, researchers need to know which part of the gene codes for exons and which for introns
what is synthetic biology
an interdisciplinary science concerned with designing and building useful biological devices and systems, it includes biotechnology, evolutionary biology, molecular biology, systems biology and biophysics. Its goal is to build engineered biological systems that store and process information, provide food, maintain human health and enhance the environment
what does the sequences of DNA found by analysis genome provide
potential building blocks for synthetic biologists to build devices
what is information storage as an example for synthetic biology application
scientists can encode a vast amount of digital info onto a single strand of synthetic DNA
what is production of medicine as an application for synthetic biology
E.Coli and yeast have both been genetically engineered to produce the precursor of a good antimalarial drug, previously only available by extracting from certain parts of a plant at particular times in its life cycle
what is novel protiens as an application for synthetic biology
designed proteins have been produced, like one that is similar to haemoglobin and binds to oxygen, but not to carbon monoxide
what is biosensors as an application for synthetic biology
modified bioluminescent bacteria placed on microchip coating glows if the air is polluted with petroleum pollutants
what is nanotechnology as an application for synthetic biology
material can be produced for nanotechnology
what is bioethics
synthetic biology raises issues of ethics and security, extensive regulations are in place after 30 years of using genetically-modified organisms, many advisory panels and many papers are written on how to manage risks. Synthetic biology isn’t about synthesising life from scratch, but the potential for new systems with rewards and associated risks to be managed.
what was jeffreys doing in 1978
locating tandem repeat sequences of DNA
what are tandem repeats
repeptitative segemnts of DNA that do not code for proteins. They may be between 10-100 base pairs long and all feature the same core sequence, GGGCAGGAXG, where X can be any of the 4 nucleotide bases.
Where do tandem repeats occur
at more than 1000 locations in the genome and, in each of these places, they may be repeated a random number of times
what are variable number tandem repeats (VNTRs)
highly variable tandem repeats
what did Jeffreys obtain in 1978
some DNA from his lab technician and her parents and analysed it.
Why was Jefferys suprised with the DNA he obtained in 1978
the number of tandem repeats showed a family resemblance, but the DNA profile for each family member was unique. He realsied that a person’s DNA profile could confirm or refute maternity or paternity
What is steps 1-2 of DNA profiling
- DNA is obtained from the individual by mouth swab, from saliva on toothbrush, from blood or hair or from bones in case of ancient remains 2. the DNA is digested with restriction enzymes, these enzymes cut the DNA at specific recognition sites, they will cut it into fragments, which vary in size from individual to individual
What is steps 3-4 of DNA profiling
- the fragments are separated by gel electrophoresis and stained. Larger fragments travel the shortest distance in the gel 4. a banding pattern can be seen
What is steps 5-6 of DNA profiling
- the DNA to which individual is being compared is treated with same restriction enzymes and also subject to electrophoresis 6. the banding patterns of the DNA samples can then be compared
What did the first method of DNA profiling involve and why was it adapted
involved restriction fragment length polymorphism analysis which is laborious and no longer used
what is now used when profiling DNA
short tandem repeat (STR) sequences of DNA are used. These are highly variable short repeating lengths of DNA, the exact number of STRs varies person to person
how are short tandem repeat (STR) sequences separated
by electrophoresis. Each STR is polymorphic but the number of alleles in the gene pool for each one is small. 13 STRs are analysed simultaneously, so although each STR is present in 5-20% of individuals, the chance of 2 people sharing STR sequences at all the loci is 1x10*18.
This short tandem repeat sensistive or not and what does this mean
it is very sensitive and even a trace of DNA left when someone touches an object can produce a result, samples must be treated carefully to avoid contamination
how long can DNA be stored for and when is it useful to do so
for many years, in unsolved crimes can be used as evidence years later
How has DNA profiling transformed forensic science
brought about convictions and established victims who were previously convicted of crimes
what are some examples of how DNA profiling has been used in forensic science
identitfy Nazi war criminals is south america, identify victims body parts after air crashes, terror attacks or other disasters, match profiles from descendants of those lost in WW1
What is a childs genetic infomation made up of and what does this mean
half mums and halfs dads genetic infomation, so half of short tandem repeat fragments come from mother and half from father. This means comparing DNA profiles of mum, dad, child can determine materinity and/or paternity
what can protein electrophoresis detect
the type of haemoglobin present and aid diagnosis of sickle cell anaemia, a varying number of repeat sequences for a condition like huntingtons can also be detected by electrophoresis
what are some limitations of analsying DNA from crime scenes
if a few cells are left behind the criminals DNA could be obtained but an innocent person could have touched the same surface and be wrongly convicted
