BIOC15 Final

Trinucleotide repeats

Expansion of repeats in a few specific genes has been associated with specific neurological disorders.

Fragile X syndrome (Fmr1 gene). Huntington’s disease (HTT gene). X-linked spinal and bulbar muscular atrophy (SMN1). Myotonic dystrophy (DMPK gene). Spinocerebellar ataxia type 1 (ataxin-1 gene). Dentatorubral-pallidoluysian atrophy (atrophin-1 gene).

Depurination

Hydrolysis of a purine (A or G)

Happens about 1000x per hour in a cell. A random base is introduced after depurination. A mutation occurs three-quarters of the time. Cells will try and repair purine but will not necessarily know where. Might put in A or C by accident (instead of G). ¼ of time no mutation, ¾ of time a mutation occurs.

Deamination

Removal of amino group from cytosine or adenine. Uracil is brought into the picture. Polymerase will think uracil is thymine.

C → U, G→T, and later on you get A

Tautomers

Identical chemical formulas, but different arrangement

C# x A, T# x G and vice versa. Can’t recognize the right base.

X-ray mutagen

Break the back bone of DNA (double-stranded breaks, can’t reintroduce). Why dentists put blanket on you. Researchers often die from cancer, not that damaging anymore.

UV light mutagen

Why it is important to maintain ozone layer. Three types of UV-light, C most powerful, doesn’t come through. Power - the wavelength. Induces the thymine dimer - polymerase gets to it and stalls. May stop and fall off. Mutational age hits. Linked to skin cancer.

Oxidative radiation mutagen

Nuclear radiation is a type, multi-generational reports link nuclear plants and atomic bomb disasters to cancer. Oxidation forms nucleotides, does replication, pairs the wrong nucleotides. GO (oxidative guanine) get’s randomly paired to A instead of to C or A to T. T now exists instead of C.

Chemical mutagen

Base analogs, chemical structure almost identical to normal base but pair with different bases. Many in the environment → 5 bromouracil. 5BU is a tautomer resembling C. Hydroxylating, alkylating, deaminating, intercalators.

Hydroxylating agents

-OH group, altering base structure. Chemicals in latex and nylon production. C gets hydroxylated and can’t pair with G, but pairs with A.

Alkylating agent

Add ethyl or methyl groups. EMA used in labs, plant systems. Try and select new mutants. How we get a lot of GMO plants.

Deaminating agents

Remove amine -NH2 groups. Nitric acid used in synthetic dyes. Changes C to be read as U. Uracil pairs like T instead of with G or whatever.

Intercalators

Insert between bases causing insertion and deletions. Polymerase has issue getting passed. Disinfectants. Even though it is considered safe now, people still wear gloves.

Alkyltransferase

Repairs guanine modification (special enzyme)

Photolyase

Repairs thymine dimers

Base excision repair

Big protein context fixes all this. Mistakes can be recognized. Mismatch pairing is recognized, glycolases remove the base that’s wrong. Nuclease that gets cut. Open gap. Synthesis by DNA polymerase, needs a big gap to do it.

Nucleotide excision repair

Response to UV exposure. Fixing thymine dimer. Nips strand around bases that were fused together, DNA ligase closes the gap.

Exposure → thymine dimer form → UvrB and C endonuclease chop strand with dimer → damaged fragment chopped → DNA polymerase fills gap → DNA ligase seals gap

Homologous recombination

Whole DNA molecule is broken. If DSB occurs during time of sister chromatid in the cell. DSB repaired uses sister chromatid as framework. Not the same as recombination itself.

Nonhomologous end-joining (NHEJ)

Most common way of fixing, where there is not a way of homologous recombination - cleans up ends

Sloppy polymerase (bacteria)

Last restort (error prone). If DNA is really damaged, when polymerase stalls and can’t continue they basically incorporate random nucleotides. As soon as they get into region of DNA.

Microhomology-mediated end-joining (MNEJ)

Last resort (error prone). When 2 strands of DNA are broken apart, a group of enzymes starts restricting till it gets to an end where it can start resection with each other. Cut back on either side till there is a compatible region (annealing, flap removal, fill-in synthesis, ligation)

Deletion to homozygosity

Lethal or harmful, depending on size

Deletion to heterozygosity

Have mutant phenotype due to dosage effects, increase risk of phenotype due to recessive mutant allele. Basically increased risk of recessive allele taking over.

Tandem duplications

Occur on same side of the chromosomes leg/right next to each other (can be same order, or reverse order). Barcode in multi-omics

Nontandem duplications

dispersed duplication. Occurs on same chromosome but not right next to each other.

Microduplications

Affect a single gene. All patients had duplications affecting the same gene. Triplosensitivity of the gene.

Pericentric inversions

Centromere is within the inverted segment

Often becomes unviable. Each recombinant chromatid has a centromere but each will be genetically unbalanced.

Paracentric inversion

Centromere is not within the inverted segment.

Is unviable. One recombinant lacks a centromere entirely, only producing half gametes.

Can act as cross over suppressors → In inversion heterozygotes, no viable offspring are produced that carry chromosomes resulting from recombination in inverted region. If you follow line of recombination, you will get two chromosomes where recombination happened. 2 centromere. 1 lacks centromere. Acentric fragment is lost.

Translocations

Two different non-homologous chromosomes. Requires DSB on both. Still have fully complemented genes, just carried through elsewhere. The effect depends on where breaks occur. Pseudo-linkage, robertsonian, aberrant crossing over.

Pseudo-linkage

What happens may not be linked on same chromosome because of translocation between chromosomes. Fusion.

Robertsonian

When you have breaks in 2 acrocentric chromosomes. The chromosome had a very small or big arm

Transposable elements

Segments of DNA that can move from place to place within a genome. Repetitive regions. Exist in all organisms. Hundred of thousands of copies per genome.

Retrotransposons

Long term null repeat regions. Within stuff, DNA transposons work differently.

Copy and Paste jump

(ERV and LTR, Non-LTR retrotransposons:

DNA carries transposons inside of a sequence, the enzymes and everything it needs to make mRNA for its business. That transcribed into DNA. Integrated in new location. Copy is reintegrated somewhere else. Target primer reverse transcription. Transcribed directly in. Result is the same. Small homology region.

Cut and Paste

DNA transposons

Don’t get copied. Homologous region don’t get increased. Excision process is not always very accurate. As you get excised, integrated elsewhere, this may disrupt gene it is integrated in.

Autonomous TEs

Non-deleted that can transpose on their own

Nonautonomous TEs

Defective that require the activity of non-deleted copies of the TE for movement

VDJ recombination intiation

RAG-1 and -2. Generally expressed DNA repair protein then carry out the joining reaction

Sanger sequencing

Chain termination method. Add dNTPs, then ddNTPs (missing OH group). Oxy-bridge can’t form and chain terminates. ddNTPs are fluorescently tagged. We only know where the terminal base is. Nested fragments. Get 5’ to 3’. Computer reads the fluorescents.

Each land displays the sequence of one DNA sample + primer. The gels are scanned and transmitted into computer - detects the lights. Produces chromatogram

Shotgun sequencing

Used to help sequence the human genome. Principles are that it breaks genome into overlapping fragments. Sequence lots of fragments randomly, assemble sequences base on overlap to form contigs.

Contigs, Issues with repeat regions - low selective powers - lose some repeats., Paired-end sequencing: sequences two DNA reads separated by an insert of a known size. Reads + inserts = fragments

Contigs

Contiguous sequence - output of sequencing is reads. You don’t go from a read to a genome. You overlap and assemble this and try to close the gap. Produce a whole genome.

Illumina sequencing

1. Sample preparation. High quality check - for purity concentration of DNA to work (using photo spectrophotometer). Adaptors with speciific sequence added to each end. Flowcell adaptor growing like grass on a lawn. First converted to a single strand

2. Cluster generation. Fragment of interest wasted away. Forms a bridge, connecting with its opposite. They match up all complimentarity.

3. Sequencing. Sequencing is repeated for the reverse of the fragment. Tagged dNTPs. Identity of last base pair added is noted until the read is done. Sequence denatures - see it on the computer. Now flip.

SMRT Sequencing (PacBio)

Very long fragment or could be whole prokaryotic genome. Must be fairly high quality. Adaptors are added on → create a circular molecule. Single molecule being sequenced at the end of the day. After denaturation, it’s synthesized all at once repeatedly to get many reads. Output is fragmented.

• Channels - ends have a DNA polymerase that is anchored to bottom of channel. Fluorescently tagged dNTPs. Detective only able of seeing fluorescent. DNA polymerase goes through single molecule. Get an output. Physical stategy.

• Reads are about 5 kb in length, up to 25 kb.

• High fidelity (>99.9% accuracy)

• Fewer reads than Illumina sequencing. Easier for assembly. Get less reads overall.

Nanopore sequencing (ONT)

Passing DNA through a membrane pore - hard to actualize and do practically. Artificial membrane. All tiny pores. Enzyme captures DNA from 5’ end, unzips it - forces single strand through. Maintained across the channel.

Batches of bases take different amount of time to pass. Amount og time is an addition point of data.

PHRED scores

Quality of each base pair. High quality - lower chance of error.

Q = -10log(E)

E is probability of an error

Assembling reads (reference assembly)

you can assemble data with or without reference. Known genome has previously been sequenced - read output. Align reads by consensus. Consensus sequences. Joined to be continuous, assembled into whole genome. Overlapped reads are joined to form a consensus sequence.

de novo Assembly

Assembly from fresh, without previous data. Algorithmic approaches. Ways of arranging big data. More likely outcomes. Know reference. You compare reads to one another. Using complex algorithms. Create into contigs. Get scaffolds. You don’t need reference but it takes longer and it is less reliable. Assemble reads using complex algorithms generating de Bruijn graphs to create contigs.

Annotation

identifying genes and other genomic elements. Compare to gene and genome sequences uploaded to public repositories

Comparative analysis

Comparing genes and genomes from different taxa. Classifying homologous genes.

• Orthologs: gene variations created by speciation events

• Paralogs: gene variation created by gene duplication events (often form gene families)

Autopolyploid

All chromosome sets are derived from the same species

Allopolyploid

hybrids in which chromosomes sets come from distinct, but related species

Amphidiploid

Has two diploid genomes, each from different parental species. Two fully different parents. Hybrid of wheat and rye.

Circus plot

Edges has different chromosomes - lines are connecting regions of homology - same on different chromosomes. says most likely have duplicated chromosomes. Through translocation - separated into different things. Used to study and identify in evolution studies.

In Situ hybridization

Multicolour banding produced by using FISH probes specific for regions of chromosomes. you can see large deletions. Detect chromosome rearrangement with this under a microscope.

Short hybridization probes

Can distinguish single-base mismatches. Hybridization of short (<40 bases) oligonucleotides to sample (target) DNAs (Allele-specific hybridization)

Mismatch - hybrid will not be stable at high temperatures

Cloning

1. Isolate DNA of interest

2. insert into vector (plasmid, bacterial artificial chromosome, yeast artificial chromosomes)

3. Insert vector into cells (use non-pathogenic E. coli)

4. Select and grow transformed cells.

Gateway cloning

Relies on base-pairs and LR clonase. With primers, you now add and then donor vector gets flipped into vector.

TOPO cloning

Comes with a backbone part of a kit and gets cut up. Backbone keeps the rest of it together. You cut out the PCR product.

Genomic equivalent

Number of clones in a perfect library

= length of genome / average size of inserts

Clone transgene

Introduce transgene into the genome of the germ cells → select for transgenic individual

Targeted mutagenesis

Add regions of homology to whatever you’re cloning, target different region of genome you’re trying to target. Gene targeting with homologous recombination. CRISPR (predominant).

In vivo gene therapy

Therapy delivered to somatic cells in the body (Ex. injected into retinal cells or inhaled into lungs)

Ex vivo gene therapy

Cells removed from body, treated, then put back in (Ex. bone marrow cells). Treats SCID-X1 (bone marrow cells)

Omnigenic model

All genes expressed in a cell can affect the expression of a given trait. Peripheral genes do not have a direct impact on expression but explain more heritability of traits than core genes do.

Core genes

The genes that make the stuff for your trait. In puberty → Gnrh1, estrogen

Regulatory genes

The geens that control the expression of your core genes. TF that bind to Grnh1

Peripheral genes

Genes that function downstream of core- and regulatory-genes.

Multi-omics

We can sequence more than just a whole gene - really finding the focus. Sequencing technology that has been created. Pull out transcription factor with sequencing. It’s DNA sequencing. But instead of doing everything, you’re choosing specific area.

FISH karyotype

Allows us to anchor our assembly to physical regions on chromosomes

In silico karyotype

Fluorescent karyotype. In metaphase, genetic markers tag. Most likely to get rearranged are coloured in a specific way. Structural origin of our shit compared to theirs