BICD 100

How much will it likely cost to sequence your genome in a few years?

$100

Annealing Temperature

- 40-60 degrees C - optimized for specific binding between primers and the templates

PCR

- Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) - The polymerasechainreaction, PCR, can produce many copies of a specific target segment of DNA - A three-stepcycle— heating, cooling, and replication— brings about a chain reaction that produces an exponentially growing population of identical DNA molecules - Taking advantage of natural DNA replication and using temperature changes to drive it forward - The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase. - Starts off quite small but we eventually have hundreds of copies - Start of with genomic DNA but we are interested in only a small part so we design primers that match up to either end of our target sequence so that our primers will anneal to each side and DNA polymerase can copy one rotate one section and we do this over and over again - leave the haystack there, don't find the needle, just make so many copies of the needle that you have a pile of needles

Visualization

- At the molecular level we can't see/ tell one protein from another so we need a way to visualize what we need/want to see • Heavy isotopes • Fluorescence • Radioactivity

Prokaryotic vs Eukaryotic Genome

- Eukaryotes often have much larger genomes and more genes - Prokaryotes have no introns in protein-coding genes (gene density is higher, genes back to back to back) - Prokaryotic has very low noncoding DNA

Genomics - techniques for sequencing individual fragments

- Sanger sequencing - Generation sequencing

A point Mutation

- a change in a single nucleotide (often a substation// misplaced base pairing) - can have a significant impact on a trait - accounts for 90% of human genetic variation - often occurs during DNA replication - can lead to the production of an altered protein - ex. sickle cell anemia

Insertions or deletions of nucleotides

- additions or losses of nucleotide pairs in a gene - may produce a frameshift - RNA is translated by the codon (sets of 3 nucleotides) - relatively small mutations

What DNA mutates?

- all of DNA mutates, mutations happen anywhere it can happen - some DNA mutates more often than other DNA - some mutations are acted on more heavily by natural selection

hierarchical shotgun sequencing

- break up DNA of the entire genome into fragments, sequence fragments and find overlapping pieces, then assemble via a computer - pretty much sequencing a genome 30 times over to account for any errors - cheap, easy, fast

What two pieces of information does each band give us? Why is there a lane for ɸX-174? What is making these bands visible?

- fragment length (shorter fragments move through at a faster rate) and brightness (tells us concentration)

Duplication

- have evolutionary distinction, this is where new genes come from - at first the gene works the same as the OG but over generations they can create new mutations and now that you have 2, one can mutate while the other stays the same Ex. venom now in snakes fangs not just in pancreas because the gene for venom was duplicated and the second one mutated resulting in a change of where the protein should be made

Alterations of Chromosome Structure

- larger mutations b/c it changes the entire gene - breaks but glue back together, however in that process they parents always glued correctly

STR

- short tandem repeats - STR analysis is commonly used as genetic markers in DNA profiling - STRs are short DNA sequences that are repeated many times in a row at the same location - Current standard for DNA profiling is to analyze 13 different STR sites - sequences of repeated base pairs of DNA, usually no more than two to six. If repeated excessively, they are often associated with neurological disorders, such as Huntington's chorea - Common ex. CAGCAGCAGCAG

SNP

- single nucleotide polymorphism - If you look at a population at a certain locus, SNPs are when that nucleotide isn't the same for everyone - Makes up 90% of the genetic variation between us

Gel electrophoresis

- sorts DNA molecules by size - If you put DNA in an electrical field, it would move towards a positive electrode (because DNA is uniformly overall negatively charged) - Negative electrode at the top, positive electrode on the bottom

SNP genotyping

- unlike Gel Electrophoresis, Southern Blot, and PCR we aren't losing at a section but rather an overall view of alleles and the SNPs - illumina and 23andMe - 99.5% identical between humans - 90% of variation is SNPs - 10 million SNPs - beads covered with tiny DNA fragments (that extend just up to the polymorphic position but doesn't include it) that have been synthesized to correspond to the DNA besides a particular SNP . DNA is denatured and anneals to the fragments they are complementary to. Then DNA polymerase will add one labeled nucleotide at each 3' end (complementary to what they have). C (green) and T (red) fluorescent differently and if both are present (heterozygous) you get a yellow fluorescent. - runs in parallel on many different beads (hundreds of thousands)

Southern Blot

- we can use this to identify whether a particular sequence of DNA happens to be in a sample - used not only to identify the quantity of a particular sequence of DNA but also RNA and protein - take genomic DNA in a southern blot and cut it up into tiny pieces, once you have these tiny pieces you run it on a gel - the point of the gel is less to determine what is longer or shorter but more to physically separate out fragments of DNA - once you have the gel you put it under some weights (you might use some suction) but more or less you have this film that when you stick it to your gel the DNA sticks to it and you bond the DNA to that membrane so you have little bits of that single stranded DNA that is stuck to that membrane in these exact positions - what you can do to identify if your strand of interest is there is to design a probe that is a short bit of DNA that is complementary to what you are interested in (for visualization you have to make sure your probe will fluoresce) - doesn't require PCR - finding a needle in a haystack

DNA is made up of

1. A phosphatase group attached to the 5' C 2. A 5 C sugar 3. A nitrogenous base attached to the 1' C (Adenine, Cytosine, Guanine, Thymine)

Rules of Nucleotide Polymerization

1. DNA/RNA double strands H bond in antiparallel conformation (5' and 3' end) 2. DNA and RNA polymerase only add nucleotides to the 3' end 3. Only RNA can begin a newly formed strand. DNA polymerase must add DNA nucleotides to a preexisting strand (effect: new strands must begin with RNA primers) DNA polymerase has to covalently bond something to something it can just toss that first nucleotide in

Alterations of Chromosome Structure Examples

1. Deletion 2. Duplication 3. Inversion 4. Translocation (between different chromosomes)

Charles Darwin, Origin of Species

1. Evolution as fact and common ancestry (pattern) 2. Natural selection drives adaptation (process)

Translation Steps

1. Initiation 2. Elongation 3. Termination

five fingers (modes) of evolution

1. Population can shrink 2. Mating based on appearance 3. Mutation in genes 4. Movement/gene glow 5. Natural selection

3 steps of evolution

1. Variation in a trait exists within populations 2. Traits are heritable (DNA) 3. Not all alleles are equally passed to the next generation

Principles of Natural Selection

1. Variation within a trait 2. Variation is heritable 3. Not all individuals reproduce equally/not all offspring survive to reproduce

How many nucleotides are in the human genome? How can we measure it (sequence, what variation exists, and what do those nucleotides mean)? How can we use that knowledge for our own benefit?

3.2 billion (enormous!) by reading DNA by manipulating cells and DNA

Promoter 5'AATGCGCTAGCG3' What is the sequence of the RNA transcript?

5'AAUGCGCUAGCG3'

Extension Temperature

72 degrees C - temperature that is optimal for taq polymerase

Denaturation Temperature

95 degrees C

DNA replication: Semiconservative model

A model of DNA replication that suggested that the DNA molecule split apart and each strand of the double helix of DNA served as a template for the new strand

natural selection

A process in which individuals that have certain inherited traits tend to survive and reproduce at higher rates than other individuals because of those traits.

Allele

A specific variant of a gene. Different individuals of a species have the same genes but may have different alleles

Chargaff's Rule

A=T and C=G

Pangenesis

An early, incorrect explanation for inheritance. Characteristics acquired during the parent's lifetime could be transferred to the offspring.

Why was natural selection not initially accepted?

Because genetics as a science didn't exist yet

Modern Biology = The Modern Synthesis

Brought together genetics, evolution, and molecular cell biology

What was the first genome sequenced? A. the human genome B. the yeast Saccharomyces cerevisiae C. the bacteriophage ΦX174 D. the bacterium Escherichia coli E. the fruit fly Drosophila melanogaster

C and D soon after

HGP vs. Celera

Celera didn't map out the genome after chopping they reconstructed the genome using just the overlaps - HGP put their map online for the public though

Fitness is a measure of an individual's

Contribution to the gene pool of the next generation

DNA structure

DNA consists of two long chains of nucleotides twisted into a *double helix* (with major and minor grooves and a 5' end and a 3' end) joined by hydrogen bonds (polar covalent) between the *complementary bases* adenine and thymine or cytosine and guanine

Which of these criteria have been used in selecting species whose genomes have been sequenced? A. The species should be relevant for human health or well-being. B. The species should have a relatively small genome. C. The species should have an important ecological role. D. The species may yield key evolutionary insights. E. All of the above.

E

Blending

Early and incorrect explanation of inheritance. Hereditary material form parents mixes together to form an intermediate trait like mixing paint - not possible because it wouldn't allow for an advantageous trait to show up

The Structure of Eukaryotic Gene

Eukaryotic genes undergo RNA processing (splicing and taking out introns), Prokaryotic genes do not // transcription and translation can not occur at the same time for Eukaryotic genes

Mircroevolution

Evolution within a species

Deletion

Ex. Williams Syndrome

The central dogma

Genes to traits. DNA makes RNA and RNA makes proteins

two base pair deletion in myostatin gene

Inactivates entire gene // extreme muscle build up

When a trait looks like it is blending...

It is likely because that trait is likely influenced by multiple genes and the environment so it looks like it is continuously varying when in actuality it's still the result of distinct genetic traits that aren't altered except by mutation from one generation to the next.

How many human populations are there now compared to 500 years ago?

Less (increase in migration)

Ribosome

Makes proteins // translation - allows for a tRNA/ anticodon (that is attached to an AA) to match up with the RNA strand

Molecular Clock

Model that uses DNA comparisons to estimate the length of time that two species have been evolving independently aka how long ago they may have shared a common ancestor - an important scale - If we look at variety of different parts of our genome in us and other species we are going to see that because of natural selection those mutation rates are a little different - genes that mutate the fastest are good for small scale, short time period questions - the ones that mutate the slowest can help us answer questions that are billions of years old

How does RNA polymerase know which is the template strand?

Promoter tells RNA polymerase where to attach and what direction to transcribe; RNA polymerase then uses the strand that complements 3' growth to be the template strand

Mendel's pea plants

Showed traits weren't blending but rather that traits were distinct. Individual has 2 allele and those alleles were passed from one generation to the next.

Sickle Cell Anemia

Single point mutation. Glu to Val

Gel Electrophoresis Steps

Take DNA samples and dump them in the dark spots, which are wells. We put it through an electrical current and then all of a sudden these samples start moving down (negative electrode at the top, positive electrode on the bottom) - On the far left bands are associated with numbers. This is the "ladder" and will be our metric for measuring distance. - doesn't require PCR

If inheritance of traits was via blending what would happen to a highly beneficial trait that initially mutates in a single individual?

That individual would produce many offspring and the beneficial mutation would eventually disappear.

Bacterial cell vs Eukaryotic cell central dogma

The central dogma is universal. At its core how DNA replication, transcription, and translation works is nearly identical in all living organisms. In general the flow of information is the same. Theses processes in both cells have many similarities and many of these similarities are at the basic conceptual core.

Huntington disease diagnosis

The more CAG repeats you have, the more likely you are to have Huntington disease

Rosalind Franklin and Maurice Wilkins

Used x-ray crystallography to study DNA structure

Thomas Morgan & Mutation

Working with fruit flies he showed that traits/genes could be tied to a location on chromosome. The discovery of genetic mutation backed up the idea of natural selection. - X-linkage in Drosophila: White eyes are mutant, red eyes are wild type

Reading a genetic code

You must start at a start codon and end at an end codon (nothing can be coded after the stop codon)

Which of the following is the best primer pair? a) AAAAAAAATTTTTTTT & TTTTTTTTAAAAAAAA b) ATCG & AGCTA c) ATCGTGTAGTCGAGCGA & ATGCGATGGGATCGTTA

a - no b/c they can attach to each other or themselves (creating a hairpin) b - no b/c not unique enough b/c it is so short c - yes b/c unique so will likely only bind in the place we want, needs to have about the same H bond so it will have the same annealing temperature

Evolution

a change in the genetic composition (allele frequency) of a population over time (across generations)

How fast can your genome currently be sequenced?

a couple hours

Population

a localized group of individuals capable of interbreeding and producing fertile offspring

Termination

a stop codon is reached

Frameshift

add or take out nucleotides that aren't a factor of 3 - relatively small mutations, like 1 or 2 or a dozen or so nucleotides

Which type of point mutations typically has the greatest positive effect on proteins function? a. silent b. conservative missense c. nonconservative missense d. nonsense e. there is no way to tell

b, c, or e

Generation sequencing

being able to run many different sequencing in parallel just like in SNP genotyping

How do we manipulate DNA?

by its negative charge, it is one of the most negatively charged things in a cell - we can use that to stick it to a filter or change its solubility

A random point mutation somewhere in the human genome is most likely to have a __________ effect. a. positive b. negative c. no effect

c - most are introns (1.5% are exons)

Missense non-conservative point mutation

causes a different AA to be coded for that is chemically different to the one that was originally supposed to be coded for

Missense conservative point mutation

causes a different AA to be coded for that is chemically similar to the one that was originally supposed to be coded for

Elongation

codon recognition, peptide bond formation, translocation

Gene pool

consists of all the alleles for all loci in a population // if populations don't or can't interbreed they don't share the same gene pool

nonsense point mutation

creates a stop codon (chain terminating mutation) that is deleterious for the cell

Which type of point mutations typically has the greatest negative effect on proteins function? a. silent b. conservative missense c. nonconservative missense d. nonsense e. there is no way to tell

d

What ingredient is not necessary for PCR? a) Template DNA b) DNA Polymerase c) dNTP's d) Helicase e) Primers

d - we use heat to unwind/denature the DNA dNTP's are our nucleotides

DNA vs RNA

deoxyribose sugar vs. ribose sugar (OH on 2'), thymine vs. uracil

Sanger sequencing

each fragment is sequenced letter by letter - take a single strand of a template, we set it up so we know the sequence at one end so we can have a primer like in PCR, instead of getting tons of identical complete copies of our fragment we get tons of different length fragments one for each possible length from the beginning to the end, we know the identify of the last complementary nucleotide because we make sure they fluoresce a different color - so now we need to figure out the order, gel electrophoresis can separate out fragments based on their length and ends up giving us a sequence - slow and expensive (can't really run in parallel on the same scale of generation sequencing)

Repetitive DNA

explains why we have such large genomes because they just copy themselves don't really do much to help you - majority of our genome

Initiation

finding the start codon

Every cell in our body...

has the same genes

H bonds between complementary bases in DNA can be broken via

heat or change in charge/pH

Genomics - techniques in assembling small sequences into whole genomes

hierarchical shotgun sequencing

Mutations are happening frequently but...

most of the times there are pathways that fix these mutations but it is a good thing it doesn't fix it all of the time so that there is some genetic variation

natural selection vs mutation increase, decrease, or either fitness: #of alleles in population

natural selection: either, decrease mutation: either, increase

If Annealing Temperature is too high?

no products made

nothing has progressed faster than _________

sequencing technology

silent point mutation

the mutation causes no change in the amino acid coded for

After Transcription and Translation

the strands get significantly smaller (even before RNA processing) in order to express some genes when needed and not express others when not needed - also information is passed on through each process

Genomics

the study of an organism's complete set of genes and their interactions - looking at all 3.2 billion nucleotides - first step is reading the sequence of a fragment // none of these technologies have the ability to read a whole chromosome so we break it up into fragments, read them, and then links them up again

Genetics

the study of heredity and the variation of inherited characteristics

Mutation

the ultimate source of genetic variation

If Annealing Temperature is too low?

too many products made

unique noncoding DNA

we don't know why we have these or their purpose

If you were homozygous for something and you isolated that one locus and ran it on a gel...

you would only see one band because your version for you father and mother are identical and the same length so they would travel the same distance and be mixed together

Ways to read alleles (measure the human genome)

• Gel Electrophoresis (STR analysis) • Southern Blot • Polymerase Chain Reaction • SNP genotyping