Cycle 11 - Genomics

Question 1

Describe the basic mechanisms of human gene therapy

Answer 1

• One way is through viral vectors to deliver out transgene into a specific tissue (ex., liver)
• Or we could remove stem cells from people, transform them in vitro, and then reinsert them (best in blood)
• Or we could use embryonic stem cells and transform them and then introduce them into a patient

Question 2

Describe CRISPR/Cas9 functioning in bacteria

Answer 2

• CRISPR is a mechanism that confers immunity from viral attack to single-celled bacteria
• Bacteria that survive the first infection are now much more resistant to a subsequent infection by the virus through CRISPR
• Each spacer in the DNA is different and each is derived from the genome of a previously infecting virus
  
  • These spacers are passed onto daughter cells (passing on of acquired characteristics!)
• Cas9 is an endonuclease that cuts DNA (not site-specific, unlike restriction enzymes)
• TracrRNA + cas9 (protein-RNA complex) form a complex
  
  • Tracer gets cas9 associated to the pre-CRISPR RNA to create the mature complexes
  • Waits for the virus to attack again, when the virus enters the cell, cas9 finds complementary base pairs and makes a doubles stranded cut to destroy the viral DNA

Question 3

Describe the basic mechanism for CRISPR/Cas9 in genome editing

Answer 3

• We make our own guide RNA that directs cas9 to any sequence we choose to make double stranded cuts
  
  • We can knock it out or install our own donor DNA (this is homology dependent repair; if you offer the system an alternative sequence it will get incorporated)

Question 4

Describe the fundamental ethical issues arising from human genome editing

Answer 4

• CRISPR babies
  
  • Gave them HIV resistance… but that’s unethical; don’t know the effects of the change when we remove CCR5 to confer resistance

Question 5

Describe the rationale and process for creating “three parent babies”

Answer 5

• The rationale is to prevent defective maternal mitochondria from being passed onto all of their children
• We take a cell from a donor and suck out the nucleus and then take the nucleus from the parents’ embryo and transfer that nucleus into the donor embryo

Question 6

Describe the basic process for cloning animals by somatic cell nuclear transfer (SCNT)

Answer 6

Somatic cell nuclear transfer

• Fetal skin cells (no epigenetic marks due to fetal)
• Get an enucleated egg cell and fuse the cells together then trick it into thinking it has been fertilized to get it to develop into an embryo
• mRNA from a histone modification gene is injected into the assembly that “messes” with epigenetic treatment (we aren’t quite sure what is happening)
• Monkey is a clone of the fetal skin cell donor

Question 7

Describe why animals won’t be the same when cloning

Answer 7

• Different epigenetic markers
• Different relationship
• Telomerase differ in size
• Mutation
• Different x-inactivation

Question 8

Describe the basic process of therapeutic cloning

Answer 8

• We take one of my skin cells and one of my eggs and enucleate it, mess with the epigenetics, and then grow my cells in a culture
• Kidney failure –> grow myself another kidney

Question 9

State the strategies for identifying essential genes

Answer 9

• Find the organism with the smallest genome (mycoplasma) and look at what genes it has
• Find genes that are expressed strongly in all cells
• Knock out genes one by one and see which ones are essential

Question 10

Describe the structural features of dideoxynucleotides

Answer 10

• Nucleotide binds to the 3’ OH
• Since it lacks a 3’OH, no more nucleotides can be attached to it and so elongation stops

Question 11

Compare the classical vs. automated Sanger sequencing

Answer 11

• Classical
  
  • The special nucleotides that block elongation of a growing strand and thus stop synthesis
  • These give a series of bands that allow us to read off the sequence (top is 3’, bottom is 5’)
  • Very slow and laborious –> expensive
• Automated
  
  • Do it all in one tube
  • Innovation and automation drop the price

Question 12

How do you read Sanger sequences?

Answer 12

• Read bottom to top
• Top is 3’ end, bottom is 5’ end
• The strand that gets the primer is the one that is being blotted

Question 13

Describe the basic mechanism of massively parallel sequencing

Answer 13

• DNA is broken up into fragments and denatured to single strands
• Single-stranded adaptors complementary bind to either side of DNA fragment
• DNA fragment binds to a glass cell by the adapter through complementary base pairing
• Primers are added, and fluorescent nucleotides are added and detected one at a time using a laser
• DNA sequences from all fragments are analyzed to determine overlaps and generate a complete sequence
• Allows up to a billion different DNA fragments to be sequenced simultaneously

Question 14

Describe the strategy underlying whole genome shotgun sequencing

Answer 14

• Many copies of the genome are broken randomly into millions of overlapping fragments
• Each fragment is amplified and sequenced, then assembled together using computer algorithm that look for overlaps
• Purpose is to sequence genomes that can’t be sequenced directly (ex., circular)

Question 15

Describe the strategies underlying metagenomics

Answer 15

• DNA extraction from many organisms in an ecosystem (snakes, toad, bacteria, viruses, leaves, algae), break it into pieces, sequence it, and then put it all together, and look for cool genes, enzymes, etc.
• “Mining the biosphere for useful genes”

Question 16

Describe the strategies for annotation of genome sequence (finding ORFs)

Answer 16

• 3 reading frames on each strand (ex., ATG, TGT, or GTC are 3 starting positions that would create the 3 unique reading frames)
• Have the computer search for ORFs
• Compare it with known databases (BLAST)

Question 17

Definition of genomics, transcriptomics, proteomics

Answer 17

• Genomics: study of whole genomes, including structures, functions, and evolution
  
  • Knock out a gene and see what it does
• Transcriptomics: study of the transcriptome (the complete set of transcripts in a cell) and how levels of transcripts vary during development, in different cell types, under different physiological conditions, etc.
  
  • Upregulate or downregulate a gene and see what happens
• Proteomics: study of the proteome (complete set of proteins expressed by a genome, -protein structure, function, location, and interactions with other proteins
  
  • Observe the function and details of the product proteins

Question 18

Describe basic mechanism of using microarrays and RNA Seq strategies for analysis of gene expression.

Answer 18

• Micro-arrays allow you to screen the expression of mRNA and compare that easily and quickly to different tissues
• Micro-arrays are solid surfaces (DNA chips)
• The black squiggles are single stranded DNA sequences
• Extract mRNA and use reverse transcription, tag it
• Then, see if it hybridizes with any DNA sequences on the chip
• End up with a scan that lights up based on whether or not an mRNA was expressed in the samples
• “Micro-arrays allow you to screen the expression of mRNA and compare that easily and quickly in different tissues”
  
  • RNA Seq strategies isolate mRNAs, convert to cDNAs, sequence cDNAs and align with genome to determine which genes are being expressed

Question 19

Describe strategies for confirming that a given ORF is actually a gene

Answer 19

• Look for the mRNA transcript or protein
• Look for a promoter or polyA clipping sequence
• Compare it with known databases to see if it matches a known gene

Question 20

State the similarities and differences between the structure and organization of genes in typical bacterial vs. eukaryotic genomes with respect to such variables as gene size, presence of introns, gene density, % coding DNA etc.

Answer 20

Bacteria have 1 circular chromosome + plasmids, eukaryotes have many chromosomes

Bacterial genes are much more densely packed, 88% coding vs human where it’s 1.2%

Bacterial genes have no introns, whereas in eukaryotes, majority of genes are introns

Genes are slightly larger in eukaryotes (humans 430 amino acids vs E.coli 330 amino acid)

Question 21

Can the coding sequence of one gene be inside the coding sequence of another gene?

Answer 21

Yes, you could have two genes on different strands

Question 22

Could one gene be coded inside the intron of another gene?

Answer 22

• Yes, you could have one gene on the top strand and a different gene in that gene’s intron on the bottom strand
• Some introns in one cell can be exons in another cell
• Ex., a gene could be inside an intron in an eye cell but inside an exon in a skill cell

Question 23

Describe the basic considerations in ethical decision making

Answer 23

1. Fidelity (are you doing what you agreed to do)
2. Beneficence (are you contributing to the overall good in the world)
3. Non-maleficence (are you not being evil?)
4. Veracity (are you being truthful)
5. Transparency
6. Autonomy

Question 24

What are protein networks?

Answer 24

• Most proteins focus on metabolic processes, cellular processes, and cell communication
• Proteins interact in complex networks
• Difficult to predict the effects of networks and cascades and how they respond to changes
  
  • “Whatever you are messing with is part of a network, and the implications of changing things are going to be very difficult to predict”

Question 25

How could I create a custom creature?

Answer 25

Create a minimal genome

Add our cool genes that we mined from the biosphere (via metagenomics)

Make a brand new organism that does that I want it do to