1.4 - Major advances in genomics Flashcards
DNA sequencing
used to determine the exact order of the bases in a strand of DNA. Can be done by detecting how the bases are added to figure out the template DNA
Sanger’s method
addition of dNTPs (the base units) extends the DNA chain, but the addition of ddNTPs cause the termination of strand elongation
Pros and cons of Sanger’s method
Pros:
- Relatively accurate
Sequencers are cheaper
Cons:
- Can only be used for short - - DNA strands of 100 to 1000 base pairs
Labour intensive and time consuming
Shotgun method
Long DNA is randomly broken into numerous small segments and are sequenced using Sanger’s method to obtain reads
Pros and cons of Shotgun method
Pros:
- Fast and relatively accurate
- Allows the sequencing of multiple DNA fragments parellelly
Cons:
- The assembly part is computing-intesive and it’s difficult without an existing genome to match to
Second generation sequencing: Next Generation Sequencing (NGS)
based on sequencing by synthesis - chain elongation without termination
An example is the Illumina method
Illumina method
The DNA is cut into millions of small fragments. The adapters are added to each end, then the sequencing library is prepared
Pros and cons of the Illumina method
Pros:
- High throughput
- Cost efficient
Cons:
- Relatively short read lengths (150-300bp)
- Large initial investment
- Problematic for long repetitive sequences at the assembly stage
De novo sequencing
the initial sequencing of an organism. It requires large computing capacity to assemble the short DNA reads into a complete sequence. It serves as a reference sequence
Resequencing
sequencing an individual of a species where a reference genome sequence is available. Faster and easier than de novo sequencing. Used to find variation among individuals
DOES NOT HAVE AN ASSEMBLY STEP. It’s replaced by mapping the fragments onto the reference genome
Annotation
Process of attaching biological information to sequences. It follows the steps of:
- Identifying non-coding regions
- Identifying genes
- Attaching biological function to the genes
Transcriptome
the set of all RNA transcripts in an individual or a population of cells. Can be used to refer to all RNAs, or just mRNA (depends on context)
2 methods of gene expression detection
- RNA sequencing (but RNAs are not stable and difficult to work with)
- Complementary DNA method (cDNA) - made using mRNA as template. All the cDNAs are sequenced using NGS
RT-PCR (Reverse transcription polymerase chain reaction)
it combines reverse transcription of RNA into DNA (cDNA) and PCR to measure the amount of a specific RNA
Fluorescence is used in qPCR (real-time PCR) to monitor the amplification reaction
Genetic marker
any altercation in a sequence of DNA or other genetic trait (gene product) that can be readily detected. They mainly consist of polymorphisms (various forms). Used to:
- Identify individuals, populations, or diseases
- Locate gene with known function
- Study genetic variation
What are isozymes and allozymes
they are proteins having the same function but differ in size, shape, and the amount of charge. They can be separated via electrophoresis. They can be detected by staining
Isozymes
different forms of the same enzyme that are encoded by different genes
Allozymes
different forms of the same enzyme that are encoded by different alleles of the same gene
Restriction fragment length polymorphism (RFLP)
cuts genomic DNA into pieces by restriction enzymes and separates the DNA segments. It helps detect genetic variation, but there are a limited number of markers
Simple sequence repeats (SSRs)
aka microsatellites. The repeats are distributed at thousands of locations over the genome. It has high mutation rates, which may change the length of the repeats - genetic variation
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Single-nucleotide polymorphism (SNP)
SNP represents a difference in a single nucleotide of a DNA sequence
Genome-wise association study (GWAS)
used to identify SNPs across genomes that are statistically associated with a particular trait. Needs large pool for SNP and phenotypic data
Genome-environment association (GEA)
studies the relationship between each SNP and environmental variables. This is more indirect, but it can find stronger relationships than GWAS
