Trimester Revision Flashcards
What is bioinformatics?
Analysing and predicting information using the structure, function, regulation and organisation of the genome and proteome.
What is an example of first generation DNA sequencing?
Sanger sequencing. Bases are assigned different coloured dye to read the DNA sequence. A cheap but slow method that uses terminator bases.
What is second generation sequencing?
High throughput method that gives millions of short DNA segments that will be amplified and reassembled.
What is third generation sequencing?
DNA up to 10,000 bases can be read in a single run without amplification.
What are molecular diagnostics?
Lab techniques that analyse the health of an individual. Can examine the molecular basis of disease to draw a medical diagnosis.
What can molecular diagnostics be used for?
Accurate, detailed, reproducible identification of the mechanisms and causes of disease as well as detection, diagnosis, prognosis and monitoring response to therapy.
- Biomarker screening and monitoring
- Pathogen detection and quantification
- Genotyping to identify mutation and forensic evidence
`PCR steps:
- Denaturation: (94 C for 15 sec-2min)
- Annealing (40-60 C, primers anneal)
- Extension (70-74 C, polymerase adds bases).
Components= template, primers, polymerase and buffer
What is a Tm?
The annealing temperature of a primer. If temperature of PCR is too high above Tm, primers can’t attach. primers should be within 5 degrees of each other and should not have complementary sequences to ensure no primer dimers are formed.
RT-PCR:
PCR for RNA. RNA will form cDNA using reverse transcriptase.
What are the levels of structure a protein?
Primary: amino acid sequence
Secondary: alpha helices and beta sheets
Tertiary: interactions between the secondary structures
Quaternary: interaction between two or more tertiary structures
Does mRNA have introns?
NO, as it has been processed.
How is mRNA translated?
Using tRNA and its anticodons that correspond to codons on mRNA and bring in amino acid.
What are the types of DNA ‘variants’ in humans?
- Single nucleotide polymorphism
- Insertion and deletion
- Structural variants (DNA >1000bp affected)
- Repeat variations (tandem repeats accounting for 45% of the genome)
Where do restriction enzymes come from?
Phage Lambda
What is unique about restriction enzyme recognition sites?
They are palindromes.
How does gel electrophoresis work and what is it used for? How is it limited?
Seperates fragments of DNA by size using an electric field and a gel matrix. When looking to identify a mutation we must know which mutation is present and it must be compatible with a restriction enzyme.
How can single nucleotide polymorphisms occur?
Through silent mutation (no change in amino acid produced), missense mutation ( the amino acid changes) and nonsense mutations (the amino acid becomes a stop codon)
How can SNP’s be detected ?
By ‘chemical cleavage of mismatch’, detecting mutations by comparing DNA to its wild type.
What is capillary zone electrophoresis and how does it work?
This is a technique that seperates molecules based on their mass/charge ratio. Electro-osmotic flow from a high voltage is applied to the capillary, causing cations of the analytes to move to the cathode. Velocities dtermined by the mass/charge ratio ( a smaller molecule with a higher charge will move faster)
What is capillary gel electrophoresis and how does it work?
This is used when charge of compounds doesn’t vary with size much. Capillaries will be coated to prevent electroosmotic flow and a polymer inside acts as a sieve for separation by size. This is done at 60 C.
What happens during Sanger sequencing?
Bases labelled with fluorophores will be added to the DNA- these will terminate the DNA chain in random places, producing fragments of different length. This can be observed on an electropherogram, and the results can be sequenced (and separated by gel electrophoresis).
What is shotgun sequencing?
This a technique used for large amounts of DNA in which fragments will be generated by restriction enzymes and cloned. The clones will be sequenced using a universal primer and be assembled into contigs by computer programs that can align ends of different reads to form a single sequence.
How does HiSeq X Ten sequencing compare to the techniques used to sequence the first human genome?
This technology can sequence 45 genomes in 1 day for $1000 each, whereas the first genome took 15 years and $3 billion. The system uses ‘massively parallel sequencing’ that sequences clusters instead of single strands of DNA. Sequencing can be done by synthesis ( as DNA is formed ) or by ligation ( forming clusters of identical DNA ). The sequence can be read from an immobilised strand on a chip.
How does Next Gen sequencing work?
Uses fluorescent dyes similar to Sanger, but doesn’t use capillary electrophoresis. Instead we use array based sequencing that processes millions of reactions at the same time. Has lower accuracy and shorter read lengths, which is counteracted by reading many copies of the same fragment.
What are the three common steps in all Next Gen Sequencing techniques?
- Library preparation: a library is created by fragmenting DNA with restriction enzymes and joining the fragments to labelled linkers (adaptors)
- Amplification of the library : by clonal amplification inside bacteria and using PCR. Resin beads will be added and DNA on the beads are complementary to sequences on the linkers , allowing binding to the beads. The fragments captured by the beads are copied by PCR.
- Sequencing: amplified DNA is sequenced by synthesis or ligation. Remaining beads put into wells on a sequencing plate with enzyme beads containing DNA polymerase and primer for sequencing. The enzyme and primer will attach to DNA on the beads and nucleotides will be added to the wells in waves of one base type at a time. Light will be produced and recorded.
How does pyrosequencing (Roche 454) work?
The 454 plate image is recorded by light given out when bases are added. More intense light means more nucleotides of the same base have been added. Does 500 megabases per run but is not reliable.
How does Illumina sequencing work and compare to Roche 454?
Illumina: cheaper, 1000 x max output over Roche, takes longer
Allows multiplexing with indexes.
- Prepare library
- Load library in flow cell and hybridize fragments to surface. Bound fragments amplified into clonal cluster by bridge amplification.
- A sequencing cycle creates a digital image of the clusters
- Alignment and data analysis is done using a computer and bioinformatics.
How does Applied Biosystems SOLiD work?
Does sequencing by ligation only, does not use polymerase, very accurate as it uses short fragments. Uses emulsion PCR using DNA bound to beads and DNA will covalently bind to surface of a slide using adaptors. Probes will bind to complimentary DNA, releasing fluorescent dye when they bind. Dinucleotides are added at each point which will be aligned in sequencing.
What are the applications of Next Gen sequencing?
- whole genome sequencing, comparative genomics, transcriptomics (RNA sequencing), amplicon (bacteria, fungi and virus) and shotgun meta genomics
Reading DNA for use:
DNA section read 100 times to identify read errors in whole genome sequencing. The reads will be mapped to a reference sequence to identify SNPs. This info can then be used for comparative genomics and for sequencing the whole genome. individual reads are put together to create a map of the whole genome.
What is transcriptomics?
sequencing of RNA to see which genes are transcriptionally active. The RNA will be converted to cDNA for the library. Used to study disease, diagnostics and treatments. Quantitative → green means more translation and red is less
What is metagenomics?
studies genetic material from the environment. Not all bacteria can be cultured. DNA from an environmental sample is extracted, cloned into a vector and transformed into E.coli. The transformed cells are screened for novel features. Contains DNA sequences for genes of microbial populations.
What is amplicon metagenomics?
looks at the gut microbiome. Looks at predetermined marker genes (short sequences). Very cheap and high throughput.
What is shotgun metagenomics?
Sequences all DNA in a mixed sample. Don’t have to sort. More accurate for functional gene analysis than amplicons. More expensive and not high throughput. DNA is extracted, fragmented, cloned into vectors and transformed into bacteria. Library is sequenced and contiguous fragments are assembled.
What is karyotyping?
Arrangement of a metaphase spread of chromosomes in decreasing length to view size, shape and number. Giesma stain used that attaches to G bands. Centromere position is on the dotted line.
