Chapter 20 - Genomics and Proteomics Flashcards
What is structural genomics?
The study of the organization and sequence of the genetic information contained within a genome
Helps generate genetic and physical maps
What are genetic maps used for and their limitations?
Genetic maps: provide the location of known genes relative to other genes (measured in map units)
Limitations of genetic mapping were overcome with the advances of molecular genetics
- Limitations: low resolution with regard to actual physical structure of chromosome/genes, doesn’t always accurately correspond to the actual physical distance between genes
What are physical maps?
Place genes in relation to distances measured in bases
Tend to have higher resolution than genetic maps
How are physical maps created?
Created through restriction site mapping
- Determines positions of restriction sites that are cut by the enzymes – gives physical landmark of where sites are in genome
Cut sample of DNA up and run it on a gel
- Number and positions of bands that you get can tell how many restriction sites are in DNA for enzyme you used - Not going to tell order of fragments
Take sample and cut it with a different restriction enzyme that cuts at different sites and run it on a gel
Then take another sample and use both restriction enzymes – called a double digest
- Run this sample on a gel and compare the bands from the single and double digests to find where overlaps occur to determine order
What are the 2 obstacles and 2 approaches of whole genome sequencing?
Obstacles:
- Large genome sizes
- Short reads and assembly can be tedious
Approaches:
- Map-based sequencing
- Whole-genome shotgun
How does map-based sequencing occur?
Take short sequenced fragments that are assembled into correct sequence where restriction sites or markers overlap
Provide known location of genes within genome at regularly spaced intervals along chromosome
Assembled into a contig – continuous stretch of DNA
How does whole-genome shotgun working?
Sequence small fragments that are arranged into correct sequence using overlap in sequence
Assembled in pretty powerful bioinformatic program
One requirement in finding overlaps is to increase coverage – average number of times a nucleotide in the genome is sequenced
- As it increases, increased confidence in assembly of genome
Can compare the genomes between species or organisms
What is metagenomics?
Genome sequences of entire group of organisms that inhabit a common environment are sampled and determined
Typically, heard of within microbial communities
- Useful for microbes that can’t be cultured
- Study communities of microbes
What is amplicon sequencing?
Sequence multiple copies of fragments from one target gene (the amplicon)
What is metagenomic sequencing?
Short sequence fragments from all DNA in population
Help see what organisms live in a studied environment
What is functional genomics?
Studies the functions and interactions of the genetic information contained within genomes
- Characterizes what genes do (especially in coding genes)
Identifies transcriptome (coding region of DNA) and proteome (proteins encoded by region)
Focuses on dynamic interaction, aspects such as gene transcription, translation, regulation of expression, and protein-protein interactions
Focuses:
- Predicting function from sequence
- Gene expression - what ways or to what degree are genes expressed
What are the 2 ways that functional genomics predicts function from sequence?
Homology searches
Protein domains
What are homology searches?
Comparing DNA or protein sequences from the same or similar species as the one you’re getting sequences from
Genes you’re looking at between individuals called homologs – genes found in different species that have evolved from the same gene in a common ancestor
- Ortholog – found in different species
- Paralog – found in the same species
Paralogs typically arise from gene duplications
Looks at different functions of genes between species
What are protein domains?
Regions of the protein that have specific shapes and functions
Common domains between species have similar arrangements of amino acids
Domains and their functions tend to be conserved over time
Can infer the functionality of proteins
What are the 2 ways that functional genomics determines gene expression?
Microarrays
RNAseq
What are microarrays?
An ordered array of known DNA sequences that serve as probes to detect the presence of complementary sequences through a process called nucleic acid hybridization
Can use this to assess the expression of genes in various tissues or under various conditions
What are the steps for microarrays?
Extract RNA from tissue sample and convert it to cDNA
- Converted through reverse transcription using reverse transcriptase
cDNA labeled with fluorescent nucleotides
cDNA is run over microarray chip
cDNA will bind to complement probes that are on chip
If they hybridize, they will emit fluorescence
What are the 3 limitations of microarrays?
Genes under investigation have to be known
Sometimes sequences will hybridize to the same probe to create artifacts
Ability to quantify degree of expression is limited
What is RNAseq used for?
Can provide detailed information about gene expression including types and number of RNA molecules produced by transcription, presence of alternatively processed RNAs, differential expression or two alleles
What are the steps of RNAseq?
Isolate RNA
Convert to cDNA
Fragment the cDNA and prepare libraries
Sequence cDNA
Assemble sequencing reads into RNA transcripts
- Can map the reads to known genes/genomes
- Can be done de novo – creating it from itself
What is comparative genomics?
Studies similarities and differences in gene content, function, and organization among genomes of different organisms
Gives us an idea of the evolution of genomes within and across species
What is single cell RNAseq?
RNAseq done in single cells
Looks at differences between two cells to see how gene expression differs
What are the 4 characteristics of prokaryotic genomes?
Single, circular chromosome
Typically small
Size of genome is variable within and between species
- Tend to see small prokaryotic genomes in very stressful or extreme environments
High association between number of genes and genome size
What is horizontal gene transfer and the 3 types?
Transfer of genetic material between organisms
Types:
- Conjugation
- Transformation
- Transduction
