1
Q
Shotgun sequencing
A
next-gen and 3rd gen sequencing techniques used
- shear DNA into short sequences
- sequence by next gen
- assembler software looks for sequence overlaps between fragments to assemble them into larger fragments (contigs)
- now the preferred way of sequencing genomes, but has problems with repetitive DNA sequences
- long-read sequences (e.g., nanopore) often used to overcome this problem
- help with assembly and alignment of short reads
2
Q
What is the ‘read’ or sequencing’ depth?
A
- the # of times a particular base is represented within all the reads from a sequencing run
- greater read depth gives more confidence a base is accurately read - ‘base calling’
- genome sequenced for 1st time: read depth is usually several hundreds - thousands
- after further sequencing (resequencing), much lower read depths are ok
3
Q
How is next gen sequencing used for transcriptomics/ gene expression analysis?
A
- isolate mRNA
- convert to cDNA
- shear cDNA
- sequence by next-gen
- bioinformatics software sorts sequences into different genes (the ‘transcriptome’)
- number of times each gene appears in sequence data = measure of degree to which that gene was being expressed in the individual/ tissue being studied
4
Q
How is the Sanger technique of DNA sequencing used in identifying species?
A
mitochondrial DNA (mtDNA) COI gene = most commonly used gene/DNA for identifying ANIMAL species
5
Q
How is next-gen sequencing used for studying microbiomes?
A
- isolate DNA from an environmental sample
- amplify microbial sequences using primers that amplify 16s rDNA gene
- sequence using next gen (e.g., illumina)
- run data through databases to see what species are present, and in what relative abundance
6
Q
How is environmental DNA (eDNA) detected?
A
- using next gen and qPCR
- isolate DNA from environmental samples (e.g., filtered water)
- using appropriate primers, informative DNA sequences can be amplified and then sequenced => species present can be identified using species-DNA databases
- particular species can also be targeted using taxon-specific primers, followed by qPCR
- can also detect eDNA in air samples
7
Q
Why do we study genetic variation at the molecular level?
A
- to determine the genetic basis of inherited diseases or phenotypic traits
- to study the relatedness of individuals or populations, and degree of intermixing of populations (population genetics)
- to identify individuals (wildlife ecology)
- parentage analysis or inferring pedigrees
- to identify criminals (forensics)
8
Q
What are minisatellites, what were they used for?
A
- used for DNA fingerprinting
- consist of 10-100 bp sequences that are repeated many times in tandem arrays
- minisatellite arrays (‘loci’) have extremely high allelic variation, due to frequent mutations involving slippage errors and/or unequal crossing over
9
Q
DNA profiling/ fingerprinting used to be done with minisatellites, what is used now?
A
microsatellites; also known as short tandem repeats (STRs) and simple sequence repeats (SSRs)
10
Q
Microsatellites
A
- like minisatellites, but shorter sequence repeats (2-5 bp)
- arrays show a lot of allelic variation, due to slippage mutations
- arrays can be amplified using PCR
11
Q
Microsatellite genotyping
A
(STR, SSR)
- PCR primers designed for flanking sequences
- primers are fluorescently labeled
- amplify products of different sizes
- separate products by electrophoresis
- genotypes identified by size of products
co-dominant:
- heterozygotes produce 2 bands, meaning both alleles are detected
- usually use same capillary electrophoresis machines used for dideoxy sequencing
- multiple microsatellites amplified at once, using primers labeled in different fluorescent colours (= multiplex analysis)
- 13 ‘standard’ microsatellite loci are used in criminal forensics => detect enough variability to distinguish all human individuals (except identical twins)
12
Q
Use of microsatellite “DNA fingerprinting” in criminal forensics
A
- PCR-based microsatellite genotyping requires only tiny amounts of DNA: ideal for forensics
- DNA-based methods have helped convict criminals and exonerated many more innocent suspects
- methods are so sensitive, though, that contamination can be a problem
13
Q
How can microsatellites and mitochondrial DNA be used to establish identities in forensic analysis?
A
- human remains after disasters or crimes are sometimes badly damaged => need genetic tools to distinguish identity
- DNA can be obtained from bones or teeth
- microsatellites (and other nuclear DNA markers, e.g., SNPs) can enable identification via kinship analysis to relatives
- maternally inherited mtDNA can also be used to establish close relationship via maternal linkages
14
Q
How can microsatellites cause genetic disorders?
A
- usually have no effect on health => are selectively neutral
- occur outside of exons (in introns or (mostly) between genes)
- a few cause diseases => all cases: loci involve trinucleotide repeats within genes or other important DNA sequences
- we all have these microsatellite loci, but healthy people have versions (alleles) with a small number of repeats
- humans with disorders (genetic) have versions with too many repeats => cause production of abnormal proteins
- ex: Huntington’s, myotonic dystrophy, fragile X syndrome
15
Q
How are restriction enzymes used to detect DNA polymorphisms?
A
(restriction fragment length polymorphism [RFLP] analysis)
- mutations either create or destroy restriction endonuclease sites
- gain or loss (restriction site polymorphisms) can be detected using gel electrophoresis
- restriction site polymorphisms most commonly caused by single nucleotide polymorphisms (SNPs)
16
Q
Single nucleotide polymorphisms (SNPs)
A
- caused by single base mutations, most common genetic variations in genome
- occurs about every 800-1000 bp in human DNA
- any 2 randomly chosen humans will have different SNP alleles at several million SNP loci
- usually di-allelic (e.g., and ‘A’ or ‘G’ at a particular position)
- SNPs close to each other on chromosome are usually inherited together (because of limited recombination) forming ‘haplotypes’
- haplotype is an arbitrarily long stretch of DNA characterized by particular alleles at the SNP positions in that sequence
- current technologies allow many SNPs to be genotyped simultaneously
17
Q
SNP chips
A
- used to genotype large numbers of SNPs
- aka microarrays; designed to allow many SNPs to be genotyped at once
- use DNA hybridization-based assay to determine genotypes at known SNPs
- have become general method of choice for rapidly screening thousands-million SNPs (loci) at once
18
Q
Genetic basis of a trait: simple vs complex
A
simple: entirely or mostly determined by one gene; ex: ear wax type
complex: most!; influenced by many genes interacting with environment
- genome-wide association (GWAS) used to find genetic links (predictors) to diseases (or traits)
- look for SNPs that have alleles correlated with presence of disease/trait
- need to survey many SNPs and many individuals
- basically look at a particular allele in people with a disease and compare frequency to people without disease
19
Q
What does CRISPR-CAS do?
A
it functions as a bacterial defence against foreign (mainly viral) DNA; designed to target specific DNA molecules, like our adaptive immune system
20
Q
What is a palindrome?
A
a sequence of DNA that reads the same from 5’ => 3’ on both strands
21
Q
How does CRISPR immunity work?
A
- spacer acquisition (‘adaptation’): a bacteriophage injects DNA which is converted to a spacer that is put in between palindromes of the CRISPR array (inserted upstream)
- expression of crRNAs: RNA transcript is made from CRISPR array (= pre-crRNA) => cut into crRNAs that each contain one spacer from a foreign organism (one palindrome and one phage RNA together); Cas9 gene is expressed to generate the Cas9 enzyme
- interference: 3 components (Cas9 enzyme, crRNA, tracr RNA) that form the effector complex; crRNA loaded into Cas9 enzyme, binds with tracr RNA, match up with invading phage genome, DNA opens up and Cas9 cuts it
22
Q
PAM site
A
- protospacer adjacent motif
- NGG (N = any nucleotide) next to spacer sequence (usually 3 bases long)
- tells Cas system to open DNA to see if there is a matching sequence (to crRNA)
- not found in CRISPR DNA array
- simple and common elsewhere
23
Q
What was the key innovation in CRISPR technology?
A
- substitution of chimeric gRNA in place of natural crRNA and tracr RNA
- chimeric = combo of different things)
- the 20-ish bases at the end of gRNA are specific to a target sequence in the genome to be edited
24
Q
Genome editing with CRISPR-Cas
A
- (s)gRNA designed to target a specific sequence in genome
- sgRNA assembles with Cas9 protein to form effector complex
- effector complex first finds a PAM, then Cas9 unwinds DNA immediately upstream of PAM => if target sequence is present, 20 b 5’ end of sgRNA pairs with it
- Cas9 makes ds-cut in genome
- cellular DNA repair mechanisms engaged with 2 possibilities: 1) broken ends can be rejoined without any template [non-homologous end joining, NHEJ], 2) broken ends can be rejoined using a template [homology directed repair, HDR]
25
Non-homologous end-joining (NHEJ)
- most common type of repair to ds-break in DNA
- no template used
- nucleotides are inserted or deleted as cleaved ends of chromosome are rejoined
- often results in insertions and deletions (INDELS)
- no INDEL => Cas9 keeps cutting until there is one
- resulting frameshift leads to non-functional alleles => gene silencing, knockout
26
Homology directed repair (HDR)
- way to repair ds-breaks in DNA
- uses same repair enzymes as in crossing over or recombination
- can use homologous chromosomes (sister chromatids) as template
- in CRISPR experiments, can inject donor DNA (aka guide DNA) at same time as Cas9-CRISPR to stimulate HDR
27
What are some advantages of CRISPR?
- cheap and easy
- targeting => can design single gRNA to target any sequence desired
- fairly specific
- INDELS created by non-homologous end-joining can create gene knockouts to determine gene function/ phenotype
- can be introduced to intact, living cells (mRNA for Cas9, or intact Cas9 protein, and sgRNA, mRNA translated by cell)
- can introduce Cas9 with donor DNA to stimulate HDR
28
Challenges of CRISPR-Cas9
- off-target effects - cleavage is sometimes not specific => modified Cas9 structure has been created to use longer (more specific) target sequence, but its slow acting; can be hard to control NHEJ vs HDR => germ-line cells have enhanced HDR => adding donor template DNA might help
- mosaicism - not all cells edited, different genomes, so get mosaic effect => delivery of Cas9 no 100% for all cells, challenge for multicellular organism; various approaches for deliver => transfection, microinjection, electroporation; embryo injections at single-cell stage
29
What are some potential uses of CRISPR-mediated genome editing?
basic research (create gene knockouts): disrupt genes to determine unknown gene functions, sometimes knocking out a gene results in a desirable phenotype
editing ('hacking') genomes to meet human needs: reversing mutations causing genetic disorders, donor organs from animals, improved farm animals (disease resistant), domestication of new plants for agriculture, 'de-extinction' (recreation) of extinct species, gene drives to eliminate insect-spread diseases
30
Gene drives
- DNA construct contains gRNA sequence, Cas9, a payload gene and flanking sequences
- once introduced as one copy, copies itself to homologous chromosome via HDR => after sexual reproduction, heterozygous offspring are converted to individuals homozygous for gene drive construct
- payload gene can spread rapidly through the population, because of non-mendelian inheritance
- best solution to malaria: make mosquitos unable to carry it
31
Why do we clone genes, gene transcripts and DNA?
- before PCR, cloning was only way to copy ('amplify') DNA sequences
- make more DNA with high fidelity for further study or manipulation (living cells copy DNA with much more accuracy than PCR)
- produce substances of scientific or commercial value from genes (ex: numerous enzymes, synthetic hormones, etc.)
- to modify genomes of plants or animals to introduce new, desired traits
32
Cloning requires a ______
vector; plasmids commonly used
33
What is a plasmid?
a stable, self-replicating molecule of circular DNA
contains:
- one origin of replication
- selectable markers to identify cells that have taken up the plasmid
- unique restriction enyme cleavage sites
34
pUC 19
typical and commonly used bacterial vector
- contains a portion of lacZ+ gene, with a restriction site 'linker' that contains numerous unique restriction enzyme cut sites
- [unique sites = found nowhere else on plasmid
- antibiotic resistance gene 'ampR' (for ampicillin)
35
How is foreign DNA inserted into a plasmid?
- cut foreign DNA with restriction enzyme
- cut plasmid with same restriction enzyme
- mix cut foreign DNA and cut plasmid DNA
- use DNA ligase to seal sugar phosphate bonds
36
How are bacteria made receptive to transformation? How do we select for recombinants?
- competent bacteria are E. coli made receptive to transformation by chemical or electrical treatment
- are also lacZ- (lack portion of lacZ gene present in plasmid)
- ligated plasmids containing DNA inserts are used to transform competent cells
- transformed bacteria are plated out on agar media containing ampicillin and X-gal
- bacteria with no plasmid do not grow (no antibiotic resistance)
- bacteria with non-recominant plasmid: produce B-galactosidase, resulting in blue coloonies
- bacteria with recombinant plasmids (lacz gene disrupted by insert): do not produce B-galactosidase, resulting in white colonies
37
Bacterial expression vectors
- make gene products
- include operon and regulatory sequences to allow expression of genes in bacteria
- good for production of many enzymes, especially those that originate from bacteria (e.g., Taq)
- not good for gene products that require post-translational modification, like many eukaryotic proteins (eukaryotic cells, like yeasts, used to make these gene products)
38
Woolly Mammoth
1. find well-preserved woolly mammoth samples
2. sequence the woolly mammoth genome
3. sequence the asian elephant genome
4. identify important cold weather genes (genes that make them well-adapted to cold environment)
5. derive cells and prepare multiplex edit designs
6. insert gene edits and create cell line
7. test the gene edits (use animal models to test traits)
8. nuclear transfer and fertilization (fuse edited nucleus of cells to an asian elephant egg)
9. implant embryo into surrogate
10. gestation and birth
39
Trans-genic Atlantic salmon
- combined growth hormone gene (cDNA) from Chinook salmon with promoter and terminator sequences for antifreeze from ocean pout
- promoter from ocean pout is constitutively active compared to GH promoter
- inject gene construct into salmon eggs
- some salmon had accelerated growth (GH gene now has constitutive promoter)
- gene construct was shown to have rearranged itself (part of promoter moved), fragmented promoter still worked just at a slightly lower level than unfragmented promoter
40
What were some measures taken to reduce the risk of the transgenic salmon escaping and interbreeding?
- grown in closed systems on land
- farmed salmon are triploid => triploid females are sterile, males can produce sperm but this risk is controlled using neomales
- neomales: sex-reversed females by methyl testosterone treatment early in development (all offspring will be female)
41
How can we use R. radiobacter to introduce foreign DNA into plants?
Rhizobium radiobacter naturally transforms plant DNA => bacteria invades plant through a wound, part of Ti plasmid is transferred to plant cell where it integrates into a chromosome => transcribed and translated to produce enzymes to help the bacteria
how we use it:
- insert foreign DNA into plasmid vector and transfer to bacteria with Ti plasmid
- plasmid vector and any foreign DNA it carries is transferred to plant cell to integrate with a chromosome
42
Introducing Bt gene to plants
- Bt toxin is toxic to insects (but not humans!)
- introduced to many plants using R. radiobacter
Process:
- clone Bt gene into E. coli plasmid to produce large amounts of the gene
- use restriction enzymes to produce DNA sequences containing various portions of Bt genes => ligated to neo gene (resistance to kanamycin; marker)
- inserted constructs into expression vector (contains promoter and poly(A) consensus sequence needed for gene expression in plants)
- put neo+BT plasmids in R. radiobacter => recombination occurs between this plasmid and Ti plasmid
- inject transformed bacteria into plant cells
- whole plants regenerated from plant cells
- if plant has kanamycin resistance, that means it took up the plasmid
BIOL 2030
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