BIOL 2030: Module 12

Shotgun Sequencing

• shear genomic DNA into short sequences

  • sequences by next gen.

• assembler software looks for sequence overlaps between fragments to assemble them into larger fragments (contigs)

• now preferred way of sequencing genomes, but has problems with repetitive DNA sequences

• long read sequences often used to overcome this problem

• genome assembly by shotgun sequencing is a big bioinformatic task

  • each segment is sequenced on average 100s of 1000s of times

Long Read Sequences

• help with the assembly and alignment of short reads

• ex. Nanopore

Read Depth

• how many times a given DNA base is sequenced in an experiment.

  • 10x read depth means each base was sequenced 10 times

• greater read depth gives more confidence a base is accurately read

• initial read depth is usually several hundreds to thousands

  • when additional copies are sequenced, much lower read depths are sufficient

Base Calling

• the crucial step in DNA/RNA sequencing where raw data (like electrical signals or light intensities) from a sequencer is translated by software into the actual sequence of nucleotide bases

• greater depth gives more confidence a base is accurately read

Shotgun Sequencing Application

• transcriptomics via next-gen

  • gene expression analysis

• indentifying species (DNA barcoding)

• studying microbiomes via next gen

• environmental DNA via next gen

Transcriptomics

• the study of the complete set of all RNA molecules (transcripts) in a cell or population of cells at a specific time, representing a snapshot of active gene expression

• The number of times each gene appears in the shotgun sequence data is a measure of the degree
  to which that gene was being expressed in the organism/tissue being studied

Identifying Species via DNA Barcoding

• though many genes/DNA sequences options, mDNA COI gene is the most widely used for identifying animal species

  • other genes are used for plants and fungi

• example: using DNA barcoding to identify sea food, or uncovering the evolutionary relationships of living and extinct species of humans

Studying Microbiomes (next gen)

• how its done:

  • isolate DNA from an environmental sample

  • amplify microbial sequences using primers that amplify 16 rDNA gene

  • sequence using next gen (ex. illumina)

  • run data through databases to see what species are present, and in what relative abundance

Environmental DNA (next gen and qPCR)

• DNA can be isolated from environmental samples

  • even in air samples

• using appropriate primers, informative DNA sequences can be amplified, and then sequenced.

  • species that are present can be identified via use of species/DNA databases

• particular species can also be targeted using taxon-specific primers, followed by qPCR

Why Study Genetic Variation

• 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

• to identify individuals

• parentage analysis

• to identify criminals

DNA Fingerprinting

• invented using minisatellite DNA

  • consist of 10-100 bp sequences that are repeated many times in tandem arrays

  • minisatellite DNA loci have extremely high allelic variation, due to frequent mutatiosn involving replication slippage errors and/or unequal crossing over

• originally relied on Southern blotting, a tedious method no longer used

  • now done using microsatellites (STRs and SSRs) which have shorter sequence repeats than minisatellite DNA. .

  • can be amplified via PCR

Microsatellite Genotyping

• PCR primers designed for flanking sequences

  • primers are fluorescently labeled, and amplify products of different size

• separate products by electrophoresis

• genotypes identified by size of products

• co-dominant

Co-dominant

• heterozygotes produce 2 bands

• both alleles are detected

Microsatellite Genotyping Equipment

• usually use the same capillary electrophoresis machines used for dideoxy sequencing

• multiple microsatellites often amplified at the same time, using primers labelled in different fluorescent colours

  • multiplex analysis

Microsatellite DNA in Forensics

• 13 standard microsatellite loci are enough to distinguish all human individuals

  • only requires a small amount of DNA

• contamination is often a problem due to sensitive methods

Trinucleotide Repeats

• in humans, a few microsatellites cause disease. in all cases, these loci invovle trinucleotide repeats within genes, or other important DNA sequences

• all humans have these microsatellite loci, however healthy humans have alleles with a small number of repeats.

  • humans with genetic disorders have versions with too many repeats. these versions cause production of abnormal proteins

• cause of”

  • Huntington's

  • Fragile X

  • Myotonic Dystroph

RFLP Analysis

• restriction fragment length polymorphism

  • use of restriction enzymes to detect DNA polymorphism

• mutations can either create or destroy restriction endonuclease sites

  • gain or loss of restriction sites can be detected using gel electrophoresis

• restriction site polymorphisms are most commonly caused by single nucleotide polymorphisms (SNPs)

Single Nucleotide Polymorphism (SNPs)

• SNPs caused by single base mutations are the most common genetic variations in genomes

  • SNP occurs every 800-1000 bp in human DNA

• any 2 humans have different SNP alleles at several million SNP loci

  • the average genome differs from standard ‘reference’ genome

• polymorphism is usually di-allelic

• SNPs close to each other on a chromosome are usually inherited together, forming haplotypes

Diallelic Polymorphism

• a common genetic variation where a specific DNA site (locus) has exactly two common forms (alleles) in a population

• due to either:

  • SNP

  • indel

Haplotype

• arbitrary long stretch of DNA characterized by particular alleles at the SNP positions in that sequence

  • physically close

  • usually inherited together

SNP Chips

• used to genotype large numbers of SNPs

• utilise DNA hybridization-based assays to determine genotypes at known SNPs

• have become the general method of choice for rapidly screening millions of loci at once

GWAS

• genome wide association

• aim is to find genetic links to diseases

• look for SNPs that have alleles correlated with presence for disease/trait

  • some diseases/traits are entirely or mostly determined by a single gene (ex. cystic fibrosis, sickle cell anemia, ear wax composition)

• need to survey many SNPs, and many individuals

CRISPR-CAS

• bacterial defence against foreign DNA now used by molecular biologists as a genetic engineering tool

  • interest in CRISPR-Cas9 type

• Clustered Regularly Inter Spaced Palindromic Repeats

  • CRSPR ASsociated proteins

• designed to target specific DNA molecules, comparable to adaptive immune systems of vertebrates

How CRISPR Works

• Targeting

• Binding

• Cleavage

• DNA Repair

CRISPR Targeting

• Scientists introduce the Cas9-guide RNA complex into a cell where it randomly associates and dissociates with the DNA. Cas9 recognizes and binds to PAM

CRISPR Binding

Once it binds to a PAM motif, Cas9 unwinds the DNA double helix. If the DNA at that location perfectly matches a sequence of about 20 nucleotides within the guide RNA, the DNA and matching RNA will bind through complementary base pairing

CRISPR Cleavage

The DNA-RNA pairing triggers Cas9 to change its three-dimensional structure and activates its nuclease activity. Cas9 cleaves both DNA strands at a site upstream of PAM.

CRISPR DNA Repair

• Cells contain enzymes that repair double-stranded DNA breaks. The repair process is naturally error-prone and will lead to mutations that may inactivate a gene. Cleaving DNA at a precise location is one of many applications of the CRISPR-Cas9 technology.

How CRISPR Immunity Works

1. spacer aquisition

   1. “adaptation”

2. expression of crRNAs

3. interference via effector complex

   1. Cas0

   2. crRNA

   3. tracr RNA

Protospacer Adjacent Motif (PAM)

• [any nucleotide]GG next to the spacer sequence

• must be present right next to the target DNA site for the Cas9 to bind and cut, acting as a critical "self/non-self" marker and limiting where editing can occur

• not found in the CRISPR DNA array

  • simple and common elsewhere

Key Innovation of CRISPR

• substitution of chimeric (combination of different) gRNA in place of natural crRNA and tracrRRNA

Genomic Editing with CRISPR

• sgRNA designed to target a specific sequence in genome

• Cas9 makes double stranded cut in genome

• cellular DNA repair mechanism engaged: 2 possibilities

  • broken ends can be rejoined without any template (NHEJ)

  • broken ends can be rejoined using a template (HDR)

NHEJ

• non homologous end joining is the most common type of repair to double strand break in DNA

• no template used, which results in INDEL mutations

• once mutation does occur, the resulting frameshifts lead to non-functional alleles gene silencing

  • knockout

HDR

• homology directed repair is another way to repair double-strand breaks in DNA

• uses same repair enzymes as in crossing over or recombination

  • can use a homologous chromosome (sister chromatids) as a template

• in CRISPR experiments, can inject donor DNA at the same time as Cas9-CRISPR to stimulate HDR

INDEL

• insertion and deletion mutations

CRISPR Advantages

• relatively cheap and easy

• can design single-guide RNA to target almost any sequence desired

  • relatively specific

• indels created by non-homologous end-joining can create gene knockouts to determine gene function/phenotype

• can be introduced to intact, living cells

• can introduce Cas9 with donor DNA to stimulate HDR

CRISPR Challenges

• Off-target effects: unspecific cleavage

• Mosaicism

Non Specific Cleavage

• challenge of CRISPR due to off-target effects

• a modified Cas9 structure has been created to use a longer target sequence, but slower acting

• can be hard to control whether NHEJ or HDR is used.

  • germline cells have enhanced HDR, adding donor template DNA may help

Mosaicism

• challenge of CRISPR

• not all cells are edited, so they get the mosaic effect

• delivery of Cas9 is not 100% for all cells, and is a challenge for multicellular organisms

  • various approaches for delivery: transfection, microinjection, electroportation

• embryo injections at single-cell stage

Uses of CRISPR

• basic research

• editing genomes to meet human desires

CRISPR Use: Basic Research

• create gene knockouts

• disrupt genes to determine unknown gene functions

• sometimes, knocking out a gene results in a desirable phenotype

CRISPR Use: Hacking

• editing, or hacking, genomes to meet human needs and desires

• reversing mutations that cause genetic fisorders

• donor organs from animals

• improved farm animals

• domestication of new plants for agriculture

• de-extinction of extinct species

• gene drives to eliminate insect-spread disease

Gene Drives

• a DNA construct, once introduced as one copy, copies itself to homologous chromosomes via HDR.

  • after sexual reproduction, heterozygous offspring are converted to individuals homozygous for gene drive construct

• in this manner, the payload gene can be spread rapidly through the population, because of non Mendelian inheritance

• could be used to insert gene for resistance to malaria, or a gene that reduces fertility of mosquito

Reasons for Cloning DNA in Living Cells

• to make more DNA with high fidelity for further study or manipulation

• to produce substances of scientific or commercial value from genes

• to modify the genomes of plants or animals to introduce new, desired traits

Cloning Vectors

• a DNA molecule, like a plasmid or virus, that carries a foreign DNA segment (gene of interest) into a host cell (like bacteria) to be copied (cloned) and amplified

• common:

  • plasmid, specifically pUC 19

Plasmid Vector

• Plasmids are commonly used as cloning vectors, as they are stable, self-replicating molecules which contain circular DNA

• contains the:

  • origin of replication

  • selectable markers to identify cells that have taken up the plasmid

  • unique restriction enzyme cleavage sites

pUC 19 Plasmid

• pUC 19 are typical bacterial vectors, as they contain a portion of the lacZ+ gene, with a restriction site linker that contains numerous unique restriction enzyme cut sites

  • unique sites= sites found nowhere else on the plasmid

Inserting Foreign DNA Sequence into a Plasmid

• cut the foreign DNA with a restriction enzyme

• cut plasmid with the same restriction enzyme

• mix cut foreign DNA and cut plasmid DNA

• use DNA ligase to seal sugar phosphate bonds

Competent Bacteria

• bacterial cells that have been treated or are naturally predisposed to take up foreign DNA from their environment

  • E. coli made receptive to transformation by chemical or electrical treatment

  • lacZ- which lack the portion of the lacZ gene that is present in the plasmid

• ligated plasmids containing DNA inserts are used to transform competent cells

Transformed Bacteria

• bacterial cells that have taken up foreign DNA, usually a plasmid, through a process called transformation, making them acquire new genetic traits like antibiotic resistance

• transformed competent cells plated out on agar media

  • bacteria with no plasmid do not grow

    • there is no antibiotic resistance

  • bacteria with non-recombinant plasmid produce B-galactosidase, resulting in blue colonies

  • bacteria with recombinant plasmids do not produce B-gala, resulting in white colonies

Bacterial Vectors Making Gene Products

• includes operon and regulatory sequences to allow expression of genes in bacteria

• good for production of many enzymes, especially those that originate from bacteria

  • Taq DNA polymerase

  • commercially available restriction enzymes

• not good for gene products thats require post-transcriptional modification, as occurs with many eukaryotic proteins

Rhizobium radiobacter

• naturally transforms the DNA of higher plants

• can be co-opted to introduce new genes to plants

  • genes such as Bt

Bacillus thuringiensis

• produces a protein, Bt toxin, which is lethal to many insects, but non toxic to humans and other animals

  • Bt gene has been transferred to many plans using R. radiobacter

Molecular Toolbox

• Restriction endonucleases(REs) repurposed as DNA scissors.

• Ligases allow DNA molecules (from different sources) cut by REs to be recombined to produce novel recombinant DNA, including
  plasmids & other vectors that can transform other organisms

• PCR is a convenient way to make modest amounts of a particular DNA sequencing, but when large amounts of DNA, or the products of genes (enzymes, proteins) are required, cloning is best.

  • also routinely used to check success of expirements

• Gel electrophoresis used routinely in combination with RE cleavage or PCR to check the success of cloning/transformation experiments.