Exam 3 Genetics biol 3010

transgenic organism

Has a gene from another individual from the same or different species

transgenesis

The intentional modification of organisms made by humans to create something that does not occur naturally

- Happens naturally: endosymbiont theory, horizontal gene transfer, viral infection, sexual reproduction

- Critical for diversification of eukaryotes and proliferation of life

What are transgenes constructed using?

Constructed and synthesized using bacteria, plasmids, and enzymes

Describe the DNA that we want to insert for genetic engineering. What is it used for?

Plasmid - circular DNA inside bacterial cells that are shared between bacteria (components differ from plasmid depending on scientist's needs)

- Used for horizontal gene transfer: opening pores so it can take in DNA from environment

Name the makeup of a plasmid and what the components do.

Origin of replication - how the plasmid gets copied inside the cell

Antibiotic resistance gene - selectable marker relevant for the cell, shows that the plasmid has made it into the cell after placing in said antibiotic

Selectable marker - relevant for organism we want to manipulate (shows the plasmid is there)

Inserted gene - shows mosaic of development and lineage (ex: GFP)

Promoter - drives expression in host organism (do not want one if we are trying to run an enhancer trap experiment - want enhancer sequences already found in host cell to promote gene expression, gives information about location and effect of different host enhancers)

Restriction site - DNA patterns that are recognized by restriction enzymes and cut so the inserted gene can be placed

Restriction enzymes - cut at restriction site in plasmid

Polylinker - stacked up compact cut sites

Primer sites - primers for PCR or molecular cloning

Origin of replication

sequence to initiate replication of a plasmid

Antibiotic resistance gene

screen whether plasmid taken up or not

Selectable marker

relevant for organism we want to manipulate (shows the plasmid is there)

Inserted gene

shows mosaic of development and lineage (ex: GFP)

Promoter region

thing that drives the expression of that transgenes (the inserted gene)

when do you not want a promoter?

do not want one if we are trying to run an enhancer trap experiment - want enhancer sequences already found in host cell to promote gene expression, gives information about location and effect of different host enhancers

Restriction site

DNA patterns that are recognized by restriction enzymes and cut so the inserted gene can be placed

Restriction enzymes

cut at restriction site in plasmid

Polylinker

stacked up compact cut sites

Primer sites

sequence plasmid and check that inserted gene is in there

Type of plasmid injection that is used in mice.

Pronuclear injection - inject directly into egg

- Linearize plasmid, cut at restriction site and inject into egg where it integrates randomly where double strand breaks are

What type of plasmid injection is used in flies?

p-element transformation

What are the two types of plasmids injected in p-element transformation?

Helper plasmid and created plasmid

What is the function of the helper plasmid in p-element transformation?

It has transposase genes that can recognize and cut P elements and also cut the host genome to make double-strand breaks for insertion.

Where is the p-element transformation injected in flies?

Into the posterior end where germ cells are located.

How long do you have to wait to see if transformation was successful in p-element transformation?

You have to wait a generation.

what does transposase do

makes double stranded breaks - recognizes p-elements ends and inserts that p-element between p-elements ends in break it made

Types of genetic engineering with transgenes

Complementation

Enhancer traps

Binary expression system

Trans-complementation

Test if "inserted gene" can rescue mutant

What is complementation in genetics?

Complementation is the process of determining whether two mutations that produce a similar phenotype are in the same gene or in different genes.

What is the first step in testing for complementation?

Map the deletion to two adjacent genes.

What do you create to test if a transgene complements a mutation?

Transgenes with wild-type copies of either gene.

What is the purpose of introducing transgenes into homozygous mutant flies?

To observe eye development and determine if the transgene restores normal phenotype.

What does it indicate if gene A transgene fails to restore normal eyes but gene B transgene does?

Gene B is responsible for the mutation.

Enhancer traps

Insert reporter gene randomly. On its own, there will be no expression but if it lands near an enhancer, it will show expression because it identifies insertions near gene of interest

- Can see where the enhancers are active based on staining of gene of interest

- Can show where enhancers are that affect expression of different genes in different locations or times

Binary expression system

Creates multi-purpose stable lines that can be used to manipulate expression, using a regulatory protein (Gal4) and a specific DNA sequence (UAS)

- Gal4 is a TF from yeast (nothing to do with fly gene expression)

Another fly has UAS sequence and target gene

- UAS is upstream activation sequence that is a binding site for Gal4

Creates Gal4 protein that recruits RNA polymerase which expresses target gene (Gene X)

- Gene X is controlled by Gal4

What are Roundup Ready crops?

Crops that are not susceptible to Roundup, a herbicide that kills weeds (glyphosate).

What is a potential concern associated with Roundup Ready crops?

The pesticide leads to potential carcinogen risks and a decline in insect populations.

What is Bt in the context of genetically modified organisms?

A bacteria that is toxic to insects, making vegetables resistant to insect damage.

What is a concern regarding resistance in genetically modified crops?

Resistance to glyphosate can develop naturally over time.

What is the purpose of growth gene modification in plants?

To promote faster growth by placing a growth gene behind a promoter that enhances its expression.

What is a characteristic of genetically modified animals related to alpha-gal proteins?

GM animals are engineered to lack alpha-gal proteins that can cause allergic reactions in humans when consuming meat.

What is the benefit of genetically modified pigs lacking alpha-gal proteins?

They can be used for organ transplants without causing reactions in humans.

What is required in the host genome to accept vector DNA?

Double strand breaks

- These happen randomly

- Repair of these can disrupt gene functions (because of in-dels)

- Can knock in genes we want during repair

How do we cut at DNA in specific spots?

Restriction enzymes

Programmable zinc-finger nucleases

CRISPR/Cas9

Restriction enzymes and pros and cons

Cut at polylinker sites to get DNA in plasmid

Pros - there are so many, found in many bacteria, and cut at many different motifs

Cons - hard to be specific, motifs are found at a lot of places in genome

Number of places a base cutter r.e. will cut? Probability?

1/4^number of bases (cutter) = probability

probability x size of genome = number of cuts

Programmable zinc-finger nucleases

Technology to make DSBs at specific sites

- Attaching restriction enzymes to zinc-fingers

CRISPR/Cas9 and pros and cons

Microbial adaptive immune system that has inherited memory (from bacteria) or from within

Pros - easy to design places and ways to target motifs for DSBs

Cons - has some off-target mutations

What does CRISPR prevent?

CRISPR (and restriction enzymes) prevent phages

- Phage infects bacteria by injecting material into host and replicating

- Phage are super ubiquitous, they are everywhere

What is included in the CRISPR locus?

tracrRNA - trans-acting CRISPR RNA

Cas9 - CRISPR associated protein 9

CRISPR locus

- Repeats - repetitive elements, prevents Cas from cutting here

- Spacers - DNA from phage genome

Steps of CRISPR in bacteria

Adaptation

Processing

Interference

Adaptation

Cas recognizes viral DNA in host and cleaves it

Cas has to look for PAM to see where to start moving the viral DNA to

PAM serves as a watch between genomic DNA and viral DNA

Cas moves the viral DNA into spacer for future immunity

Different species have different CRISPR/Cas9 and different PAM sites they recognized

Processing

CRISPR array is transcribed to pre-cRNA

- Cas is transcribed and translated, and tracrRNA is transcribed

- tracrRNA binds to Cas and makes RNA complex that binds to pre-crRNA

- RNase cleaves double stranded RNA and makes independent cRNAs with spacer between partial repeats

Interference

Transcripts bind with Cas proteins to form a complex that recognizes viral motifs and provides resistance, causes phage genome to be cleaved quicker than during adaptation

- Cas9-tracrRNA complex binds to individual crRNAs

- Spacer complementary to viral genome and complex binds here

- Cas9 cuts at PAM site a few bp away (protospacer adjacent motif, DNA 3' to spacer) and the viral DNA is degraded

What is the main difference with CRISPR in eukaryotes?

The main difference of this process is the addition of nuclear localization signals in eukaryotic CRISPR.

CRISPR in eukaryotes steps

Design sgRNA - single guide RNA, fusion of spacer and tracrRNA

- tracrRNA is complementary and binds to Cas9

- sgRNA is used as a guide for Cas9 and brings it to the target spot

- Spacer is complementary to target sequence

- PAM site is adjacent

Cas9 cuts RNA for a double strand break

What is one method of repairing double-strand breaks (DSBs) made by Cas9?

Non-homologous end joining (NHEJ) can make small in-dels.

What happens during the NHEJ repair process by Cas9?

CRISPR keeps cutting until an in-del forms.

What is another method of repairing double-strand breaks (DSBs) made by Cas9?

Homologous recombination (HR) involves co-injecting another bit of DNA that is homologous to the cut sites.

What can be included in the DNA injected during homologous recombination (HR) to repair mutations?

The middle can be a knock-in to repair mutations.

Applications of CRISPR/Cas9

Somatic gene therapy

Disease eradication

Examples of somatic gene therapy and CRISPR/Cas9

It makes a tissue functional

Ex) Leber congenital amaurosis (hereditary blindness)

- Caused by mutation that splices intron into translated bit

- CRISPR remove splice acceptor

Ex) sickle cell anemia

- Can transplant knock-in of disease causing allele

What is the purpose of using CRISPR/Cas9 in disease eradication?

To kill the vector of disease using a mutagenic chain reaction.

What components are included in the plasmid for CRISPR/Cas9?

Homology arms (H1 and H2), Cas9, gRNA, and a payload gene.

How does Cas9 achieve site-specific DNA cleavage?

Cas9 with gRNA recognizes, binds, and cuts the DNA sequence to create a double-strand break (DSB).

What is homology directed repair (HDR) in the context of CRISPR/Cas9?

The process by which a double-strand break (DSB) is repaired by inserting Cas9, gRNA, and a payload gene.

What role do H1 and H2 play in the repair process of CRISPR/Cas9?

They serve as templates for the repair of the double-strand break (DSB).

What happens if an individual is heterozygous for insertion in CRISPR/Cas9?

CRISPR/Cas9 cuts and turns them into a homozygote.

What is the effect of CRISPR/Cas9 inheritance on progeny?

Progeny will inherit the CRISPR/Cas9 modification.

How does CRISPR/Cas9 affect gene spread in a population?

It causes the gene to be spread rapidly through the population.

what does cargo gene do?

cargo kills x-bearing sperm, therefore offspring are male biased

Example of disease eradication. What does the payload gene do?

Flies and gene driven inheritance

Normal - hetero x wt homo

- Hetero spreads slowly

Gene drive inheritance - change all to homo

- Mutation spreads rapidly in population because gene is always inherited

Payload gene can kill X-bearing sperm because it cuts DNA in a cluster that is only found in X chromosome

- All homozygous offspring will be males

- Knocks back population, and since only female mosquitoes bite, it reduces the number of insects that can bite humans, so malaria is prevented

A genome consists of ______ nts.

3,000,000,000, half from mom and half from dad

What is the deal with genome size across different species?

Genome size spans a huge range. The coding sequence does not change, only the extent of noncoding DNA

What is a caveat to determining the sequences of DNA?

Only 750 bp can be sequenced at a time in a single reaction.

What are two methods to prepare for DNA sequencing?

Molecular cloning

PCR

What are the steps to molecular cloning?

Cut DNA to fragments with restriction enzymes

Isolate fragments based on size

- Gel electrophoresis: larger DNA moves slower

- Compare to template

- Patterns are different because it is based on how close or far restriction sites are

Perform gel excision and melt gel to get DNA

- Use sticky ends to get a single piece of DNA into a vector

- Ligate DNA and fragment into vector

then Transformation

what is transformation?

mix plasmids with bacteria, takes it up and amplifies it into vector

Plate the mixed cells and put an antibiotic on it so only the ones with the plasmid survive

Can use known primer sites to do Sanger Sequencing

Sanger Sequencing

Using ddNTPs to stop elongation

Need KNOWN primer sites

What are the steps of PCR?

Denature DNA into single strands

Anneal primers

Amplify DNA by generating a new double strand fragment

- Enzyme is from hot springs

- Creates an exponential growth of DNA

Sanger sequencing can be performed after

What are two ways to sequence DNA?

Sanger sequencing

Illumina

What are the steps of Illumina sequencing?

Fragment DNA using restriction enzymes and attach adapters on to the ends (bits of DNA that have sequence needed to bind flow cell)

- Adapters have index sequences so samples from different individuals can be mixed

Do PCR to amplify library, then denature to form single strands and wash library over flow cell

Perform bridge PCR to create double strand and cause DNA molecule to bend over to create cluster of clonal DNA

- Creates double strand, denature, amplify, repeat

What is sequencing-by-synthesis? What type of sequencing is it?

Growing strands nt by nt to determine sequencing

- It is paired-end sequencing - read both ends of fragments

- Maximum read length is 150bp

- Index in the adapter is also sequenced, allows deconvolution of sample

When is the best time to use molecular cloning? PCR?

Molecular cloning - useful when you don't know anything about a sequence because you can use known primers in plasmid to prime DNA

PCR - useful for amplifying a piece of DNA from a genomic DNA (need to know primer sites)

What is the difference between Illumina sequencing and sanger sequencing?

Sanger - 750 bp fragment, costs $3-8. Plasmids and cloning used, uses ddNTPs

Illumina - 300 bp fragment, 20 bil costs $2,000. No ligation into plasmids, no cloning, high throughput, uses reverse ddNTPs

What is the difference between illumina and shotgun sequencing?

illumina has no ligation into plasmids, no cloning and it uses reversible de-terminator nucleotides

How can genome construction issues be fixed? What is the next issue?

Genome construction issues can be solved if DNA fragments overlap, but the issue is that there are lots of repeated sequences, so it is hard to what order they go in

Why is Illumina not good for assembling genomes?

Illumina/Next-Generation sequencing is good for generating short fragments but bad for assembling them into genomes because of repetitive elements (simple repeats, transposable elements)

What two things are needed to assemble DNA sequences into genome sequences?

Plasmid library - short DNA fragments for shotgun sequencing; restriction enzymes cut often

BAC library - has large bits of DNA inserted into vectors that are taken up by E. coli; restriction enzymes cut less frequently; have to sequence a special way due to their length

How can you sequence BAC libraries?

Paired-end sequencing

Selective BAC sequencing

Paired-end sequencing

Can use vector's known primer sites to do Sanger sequencing of the ends

Look for overlaps between paired-end reads

Can assemble order of BACs, then use shotgun sequencing to fill in gaps

Selective BAC sequencing

Take BAC clones and hybridize to fluorescent molecules THEN hybridize each to a metaphase chromosome

Choose individual BACs that were partially overlapping and perform shotgun sequencing independently on each BAC

Independently assemble short plasmid reads within a single BAC clone because they knew where they came from and where they were

What causes overlapping fragments during digestion?

Due to decreasing the duration of time of the restriction enzyme

Incomplete digestion makes libraries with overlaps because not all cut sites are found

What is genome annotation?

Genome annotation is how you turn a genomic sequence into something usable by inferring function and presence of genes

How can you annotate the genome?

Identifying ORFs to see if transcript will make a protein sequence

Looking at conserved DNA sequences across species to infer presence of genes

Sequencing cDNA

Finding ORFs

Since genes can be encoded on either strand of the double helix, there are 6 possible ORFs

Divide DNA into all possible contiguous triplets

Where codon starts differs, and stop codons also differs

- Stop codons happen every 21 triplets (43/3)

Continuous stretch of codons means there is a gene

What are genes/exons and their cis-regulatory elements?

They are functional pieces of DNA that are more often conserved than intergenic sequences.

What are homologous DNA sequences?

They are stretches of DNA shared between species due to descent from a common ancestor.

How can homologous DNA sequences be identified?

By performing DNA alignment.

What is cDNA?

Complementary DNA reverse transcribed from mRNA

What is the purpose of cDNA sequencing?

We are trying to turn mRNA to double strand DNA

cDNA sequencing steps

Extract tissue from cell and extract mRNA from tissue

Use shared poly-A tail to prime first strand synthesis of DNA

- Add poly-T primers that bind to the poly-A tail

- Reverse transcriptase performs single stranded DNA synthesis to create a cDNA-mRNA hybrid

-Denature the hybrid and add enzyme to degrade RNA only so there is only a single strand of cDNA left

- Turn single strand cDNA into double strand cDNA

- 3' end of double strand folds back to create a self-primed hairpin so it can start second strand synthesis

- Can sequence the double strand cDNA multiple ways (cloning, Sanger, Illumina)

How can you identify function of genes after finding them?

Generating and sequencing cDNA libraries from different tissues

abundance tells you it is important to that tissue function

why do cDNA have uneven coverage?

they are purely exons