genetics lab midterm

micropipette usage

p1000- 010 = 100 microliters

p100- 010 = 10 microliters

p10- 010 = 1 microliter

purpose of the mismatch repair pathway

a quality control mechanism in DNA replication

corrects most mismatches (incorrectly paired bases) that escape initial proofreading

decreases likelihood of cancer and disease

function of MSH2 in mismatch repair pathway

mismatches recognized by MutS protein (AKA MSH2)

section of incorrect DNA is removed and filled in

Lynch Syndrome is an autosomal dominant disease. What does that mean?

only 1 copy of the mutated gene is needed to cause an individual to be affected

if parent carries a mutated gene, they have a 50% chance of passing it onto their child

when only 1 copy of the gene needs to be mutated in order to have a disease

Lynch Syndrome is often caused by germline mutations. What does that mean?

germline mutation: inherited changes in genes present in egg or sperm cells that are passed to offspring

occur spontaneously

Why do mutations at the MSH2 gene cause Lynch Syndrome?

many of the genes associated with lynch syndrome are associated with mismatch repair

mutations at MSH2 gene increases the frequency of mistakes during DNA replication

increase in mutation frequency can cause cancer and disease

why are model organisms used in research and what are they

non-human species used in lab to help understand biological processes

genetically similar to humans

easy to maintain and reproduce (short generation times, large offspring #)

well-studied genome

decreased complexity

model organisms examples

E. coli

S. cerevisiae

C. elegans

D. melanogaster

common characteristics of model organisms

genetic similarity to humans

easy to maintain and breed in captivity

short lifespan

genome has been sequenced

genes can be modified

missense mutation

DNA mutation where the codon for amino acid is changed into a codon for a different amino acid

can change protein function and cause disease or have no impact

how do you write the nomenclature for a mutation

letters on the outside, numbers on the inside

initial amino acid first, location second, new amino acid second

what does it mean for a protein to be G47P

Guanine was changed to a proline at amino acid position 47

pathogenic vs benign mutation

benign: mutation does not cause disease

pathogenic: mutation causes disease

assigned mutation of interest (letters and numbers for human and yeast) and why are we studying this mutation

human: P670

Yeast: P689L

these mutations were identified in real patients with lynch syndrome and we want to discover if this specific mutation was what caused lynch syndrome

why were yeast cells used

cheap and easy to work with

small genome

yeast MSH2 gene is 73% similar to human MSH2 gene

overall goal of mismatch repair project

determine if the introduced mutation of interest is benign or pathogenic

purpose of the protein alignment in mismatch repair project

to identify the equivalent missense mutation in the yeast MSH2

what does an asterisk mean on an alignment

it means the amino acid is the same in both sequences

CRISPR/Cas9 and Cas9 function

generate targeted genetic modifications into genomes of living organisms

Cas9 nucleases generate double strand break at a location specified by gRNA

repaired by homology directed repair or non homologous end joining

structure/function of gRNA

recognizes specific DNA sequences in living organisms and bind to Cas9

How does CRISPR/Cas9 insert a new DNA sequence into the chromosome of a living cell?

donor DNA template with intended change is introduced through homology directed repair

What is the function of the CRISPR/Cas9 system in the mismatch repair project?

we will be inserting the mutation of interest into the yeast genome using CRISPR/Cas9 to study its impacts

What is the function of the donor DNA in the mismatch repair project?

DNA made in a lab that is inserted into cells to promote homology directed repair

Why was the pML104 plasmid used in the mismatch repair project?

it contains the Cas9 gene, a gRNA spot, an ampicillin resistance gene, and a bacterial origin of replication

What is the purpose of restriction enzymes and sticky ends in DNA cloning?

restriction enzymes: proteins that cut DNA at specific sequences

sticky ends: generated by some restriction enzymes, short DNA segment with unpaired bases that allow DNA segments with complementary sticky ends to pair with each other

in cloning: plasmid is cut with restriction enzymes to generate sticky end and a gRNA sequence is mixed and DNA ligase is inserted into the plasmid

What is the purpose of DNA ligase in DNA cloning?

used to generate a phosphodiester bond and join the DNA segments to produce a stable recombinant plasmid

What is a recombinant plasmid?

genetically engineered circular DNA molecule that carries the DNA inserted into the plasmid

used to transfer genes into host cells

What is the association between DNA cloning and a recombinant plasmid?

a specific gene is isolated and inserted into plasmid to create recombinant plasmid and then transformed into the host cell

Why was DNA cloning used in the mismatch repair project?

to insert the MSH2 gRNA into the plasmid

What is a transformation?

insertion of foreign DNA (usually a plasmid) into a competent bacterial cell

What are competent cells?

have a leaky plasma membrane and are able to take up foreign DNA

What was the purpose of performing a bacterial transformation after DNA cloning in the mismatch repair project?

to insert the plasmid into the host bacterial cell and make more copies

What was the purpose of adding ampicillin to LB plates?

the plasmid contains the Amp resistance gene

only 1 out of every million bacterial cells will have the plasmid

plating all the cells on a petri dish with ampicillin will only allow the plasmid to grow due to its resistance

all other cells will be killed

Is it possible for gene editing to occur in bacterial cells that have the pML104 plasmid? Why or why not?

yes because we did so in this project

Why is the pML104 plasmid always copied when a bacterial cell divides?

the pML104 plasmid contains an origin of replication so it is copied multiple times when the bacterial cell divides

What was the purpose of the miniprep procedure in the mismatch repair project?

to isolate the plasmid from the cultured bacterial cells

involved:

1. lysing bacterial cells to release plasmid DNA

2. binding plasmid dna to spin column

3. elution of plasmid dna from spin column to microcentrifuge tube

Why is isolated DNA measured with a spectrophotometer?

it gives us information about the concentration and quality of DNA

concentration gives us the amount of DNA and protein in the sample (you want a alrge DNA amount and a small protein amount)

What is the purpose of the 260/280 ratio?

determines protein purity

want a ratio of 2:1

What was the purpose of the plasmid sequence alignment in lab 6?

to determine if the gRNA sequence was cloned into the plasmid

Describe the two sequences that were used in the plasmid sequence alignment in lab 6.

the plasmid sequence (with the inserted gRNA) and the gRNA sequence designed

What is the main difference between deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTP) in Sanger sequencing?

dNTPs: have a 3’ OH group where a phosphodiester bond can be added in the 5’ to 3’ direction

ddNTPs: no 3’ OH so when they are incorporated into DNA, they cannot be added to 3’ end which results in chain termination

What do peaks represent in a chromatogram?

a fluorescently-labeled ddNTP that has been detected at the end of a DNA fragment

What was the purpose of the yeast transformation in the mismatch repair project?

to transform the plasmid and donor DNA into the yeast cells

What two pieces of DNA were transformed into yeast cells? What was the function of each piece of DNA?

pML104 plasmid: carried the donor DNA, carried origin of replication to allow it to transform

donor DNA: carried the mutation

Why were BY4742 cells used in the yeast transformation?

they cannot grow on plates that lack uracil because uracil cannot be synthesized by the cells

yeast cells with the plasmid are able to synthesize uracil so they can grow

Why were -URA plates used in the yeast transformation?

they lack uracil

they select for cells with the plasmid

Why was chromosomal DNA isolated from yeast in the mismatch repair project?

to confirm that the mutation of interest was generated in the yeast

What was the source of the template DNA used in PCR?

the source was the yeast

What is the purpose of the polymerase chain reaction (PCR)?

to amplify the region that was targeted by the MSH2 gRNA so that the MSH2 gene can be sequenced (sequencing determines if the mutation of interest was generated in the cells)

What are the three steps of PCR? What happens in each step?

1. heat the reaction so the DNA template denatures into 2 single-strand DNA pieces

2. reaction is cooled to allow primers (short single-stranded segments of DNA) to anneal to each strand of target DNA

3. extension of the new DNA by DNA polymerase

What is the main function of primers in PCR?

to specify which region of the DNA template will be amplified

What is gel electrophoresis and why was it used in the mismatch repair project?

uses electric current forces to promote the migration of DNA fragments through a gel material (flows to the positive electrode due to its negative charge)

shorter DNA fragments move at a faster rate than longer ones

used: to determine if PCR was successful (there should be 1 band in the agarose gel)

What is the purpose of the molecular weight marker in gel electrophoresis?

has DNA fragments at known sizes so it helps determine the approximate size of the DNA fragment generated through PCR

What is the purpose of ethidium bromide in gel electrophoresis?

a fluorescent dye

can nestle between base pairs of DNA so under UV light, you can see where there is DNA

What was the purpose of the PCR sequence alignment in lab 9?

Describe the two sequences that were used in the PCR sequence alignment in lab 9. What are these two DNA sequences and why were they used in the alignment?

Describe how the canavanine assay was used to assess the effect of your mutation?

What are the differences between the positive and negative controls?

Why were the positive and negative controls used in the canavanine assay?

Was your mutation benign or pathogenic? You should be able to explain this answer with the results from the canavanine assay.

List at least two other cancers associated with MSH2

Do cancers associated with MSH2 have better or worse survival rates?

Which domain is your mutation in? What is the function of this domain?