✨Module 6: Manipulating genomes Flashcards
What are the 2 types of DNA sequencing?
Sanger sequencing
High throughput sequencing
DNA sequencing method/chain termination/Sanger sequencing technique!
1). Single stranded DNA for sequencing is mixed with DNA polymerase, DNA primers, excess free nucleotides ACTG, and terminator bases, each with a coloured fluorescent tag (ACGT). Test tubes are placed in a thermocycler and PCR begins.
2). Primer anneals to the ends of the single stranded template.
3). At optimum temp, DNA polymerase brings complementary bases to the single stranded DNA template.
4). At any time, DNA polymerase can insert one of the terminator bases by chance which results in the termination of DNA replication as no more bases can be added.
5). As the terminator bases are present in low amounts and are added at random places, this results in many DNA fragments with varying length. After many cycles, all possible DNA chains will be produced.
6). DNA fragments are separated according to length by gel electrophoresis. Lasers detect the fluorescent tags on the terminator bases and learn the order of bases in complementary DNA. From this the original strand can be deciphered.
Because each of the test tubes only contains one type of terminator base, it is possible to know what the terminal nucleotide of each fragment is.
Explain why terminator bases are so important in the Sanger method and in the modern high-throughput sequencing methods.
=> All possible length DNA fragments are synthesised.
=> Having different coloured fluorescent tags attached to 4 different terminator bases allow us to work out the original sequence of DNA.
=> High throughput sequencing is much more complex and rapid, but relies on terminator bases to terminate chains in final stages.
What is high-throughput sequencing? (don’t need to know the details).
Multiple DNA sequencing technologies that allow simultaneous sequencing of multiple DNA strands.
High throughput methods are rapid and so produce large datasets very quickly.
Why is it faster to sequence genomes now than in the olden days?
Originally, each stage was carried out by hand in the lab. But modern techniques involved machines and many DNA fragments could be processed at once.
Discuss how DNA sequencing has changed the ways in which we identify species and our understanding of evolutionary relationships.
Before, species identification was done by observation of anatomical and physiological features. With DNA sequencing, genome similarities are examined and comparisons made to standard species genome, which is much more accurate.
Evolutionary relationships - DNA sequencing looks at the difference in mutations between species. By calculating average mutation rate, you can calculate when two species diverged.
Define genome.
Contains all genes in an organism.
What is the human genome project?
An international and collaborative research program that collects DNA samples from many individuals of a species and are used to create a reference genome. More than 1 individual is used, as one individual may have mutations in DNA sequence.
How can sequencing DNA determine protein sequences?
The genetic code can be used to predict the amino acid sequence within a protein. Then they can predict how the polypeptide will fold into its tertiary structure.
Describe the function of spliceosomes.
Enzymes present in eukaryotic nuclei that remove the introns from the mRNA and fuse the exons together before translation into polypeptide.
The spliceosomes may join the same exons in a variety of ways, which would code for different amino acids, different proteins, different phenotypes.
Describe bioinformatics.
Involves storage and analysis of data (in large databases) such as DNA/RNA sequences, genotype-phenotype relationship. It allows scientists to analyse these large amounts of data generated during sequencing of billions of base pairs.
Bioinformatics allows for comparisons with genomes of other organisms. Can identify similarity between organisms to see how closely related they are => evolutionary relationships.
Data is displayed in ways that make sense and help identify patterns.
How can bioinformatics be used to investigate:
1. Genetic variation
2. Evolutionary relationships
3. Genotype-phenotype relationship
4. Epidemiology
- Many individuals of same species have their genomes sequenced and compared. A large difference in one organism means they have high level of genetic variation.
- Individuals of different species have their genome sequenced. Species with a small difference share more recent common ancestor.
- Involves ‘knocking out’ different genes/stopping their expression and observing the effect it has on the phenotype of an organism.
- Genomes of pathogens can be sequenced and analysed to aid research and disease control. Can produce antigens for vaccines.
Describe computational biology.
Uses the data from bioinformatics/different databases to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances. Working out the 3D structure of proteins, sequencing billions of base pairs.
Explain how analysing the genomes of pathogens helps.
=> Doctors can identify antibiotic resistant bacteria, ensuring antibiotics are only used when they’ll be effective and preventing spread of antibiotic resistance.
=> Doctors can track the progress of an outbreak - each strain of a pathogen has a slightly different genome and so can be accurately identified by DNA sequencing, which means place of origin/individuals with disease can be identified.
What is synthetic biology? Describe the different techniques of synthetic biology.
A recent area of research that aims to:
=> Create new biological parts and systems that already exist in nature.
=> It goes beyond genetic engineering, as it involves large alterations to an organisms genome.
=> Synthesis of new genes to replace faulty genes e.g. in cystic fibrosis.
Polymerase chain reaction/PCR purpose.
A small piece of DNA is amplified many times to produce large amounts of DNA or RNA. It is used for DNA profiling/criminal investigations and genetic engineering. It is known as in vitro (happens outside the organism) method of DNA amplification.
Describe some uses of DNA profiling and the benefits.
=> Identify criminals to prove innocence or guilt. PCR is performed on traces of DNA left at crime scenes obtained from skin/hair. This DNA profile is compared to a sample taken from the suspect.
=> Determining paternity.
=> DNA profiling also identifies individuals who are at risk of developing particular disease. Certain non-coding microsatellites have been associated with increased risk.
=>Tiny amounts of DNA can be used.
Discuss the limitations of DNA profiling.
=> Could ignore other evidence in criminal cases and mistakes could be made.
=> Contamination of samples with DNA from other organisms.
What is an intron?
Large non-coding region of DNA that is removed from mRNA before it is translated into a polypeptide chain.
Explain how PCR works.
- Reaction mixture is set up with DNA sample, free nucleotides, primers and DNA polymerase.
- DNA mixture is heated to 95 degrees C to break the hydrogen bonds. DNA polymerase doesn’t denature at this temp - many cycles of PCR can be carried out without having to use new enzymes each time.
- Mixture is cooled to 50-60 degrees so primers can anneal (bind) to the ends of the strands.
- Reaction mixture is heated to 72 degrees C, so DNA polymerase can work in optimum temp. The DNA polymerase lines up free DNA nucleotides alongside each template strand. Complimentary base pairing means new strands are formed.
- Two new copies of the fragment of DNA are formed and one cycle of PCR is complete, then the cycle starts again. Mixture heated to 95 degrees C and this time all 4 strands are used as template strands.
Each PCR cycle …
Doubles the amount of DNA. 1,2,4,8 strands.
Explain how you can use restriction enzymes to get DNA fragment by PCR.
Some sections of DNA have palindromic sequences of nucleotides. Have antiparallel base pairs (base pairs read in opposite directions).
What are primers in PCR?
Short sequences of single stranded DNA that have a complementary base sequence to the DNA sample being copied. They bind to the ends. They define the region that is to be amplified by telling DNA polymerase where to begin building new strands.
What is special about the DNA polymerase in PCR?
The enzyme used to build the new DNA/RNA is called Taq polymerase as it comes from thermophilic bacterium. This means it won’t denature at high temps involved in first stage of PCR. Also so its optimum temp is high enough to prevent annealing of DNA strands that haven’t been copied yet.
