Lecture 5

Question 1

Conjugation

Answer 1

Transfer of DNA from 1 bacterium to another through a pilus

Question 2

Transduciont

Answer 2

Transfer of DNA to a bacterium via a virus

Question 3

Transformation

Answer 3

Transfer of DNA to a bacterium via DNA uptake from surroundings

Question 4

Types of Transformation

Answer 4

1. Natural - Uptake of DNA fragments from medium surrounding a bacterial cell; only found in certain species (Pneumococcus, Hemophilus) However, does not generally occur in Escherichia coli; cells that can take up DNA are said to be competent; Tranformation typically occurs during exponential growth phase, not efficient in stationary phase cells.
2. Artificial - chemical and electroporation

Question 5

Basic steps to make compotent cells

Answer 5

1. grow up cells such that they are in exponential phase = OD₆₀₀0.6 - 0.8 A
2. collect cells by centrifuge
3. wash cells and treat cells with appropriate solution: Chemical = treat with CaCl_2. Electroporation = treat with glycerol (non - ionic) so charge goes through the cell and not through the media.

Question 6

Chemical Transformaiton

Answer 6

1. Add plasmid 4°C for 30 minutes with Ca²⁺competent cells
2. Heat shock 42°C for 1 minute
3. Recovery = 37°C for 60 minutes with added broth
4. Spread onto selective agar 37°C for 16 hours

*It is thought that the Ca²⁺ combined with the heat shock causes damage to the cell wall, allowing DNA to enter the cell

*Typical efficiency = 10⁵ - 10⁷cells/µg DNA

Question 7

Electroporation

Answer 7

1. Cells resuspended in glycero are mixed with DNA ina cuvette (with metal electrodes)
2. Cells are then subjected to a high voltage pulse (2.5kV), beleived to create pores in the cell membrane in which DNA can enter. Pulse may even push DNA( - charged) into the cell due to the non-ionic surroundings (glycerol).

*Typical efficiency = 10⁷ - 10⁹ cells/µg DNA

Question 8

What is the point of mutagenesis?

Answer 8

It is not enough to have cloned a gene or DNA of interest: the ultimate goal is to study the function of thes gene/DNA in the cell. To do this, it is important to alter the funciton of the gene (by mutation) and ask “what happened to the cell?” Prior to recombinant DNA tech, it was only possible to study mutations in DAN that were found to occur ‘naturally’ with the ability to clone DNA and perform restriciton digests and PCR. We now have the ability to make virtually any mutation we desire

Question 9

4 Types of mutations

Answer 9

1. Silent
2. Missense
3. Frameshift
4. Nonsense

Question 10

Silent mutation

Answer 10

A mutation in the DNA that does not result in a change in the amino acid sequence fo teh encoded protein (CAT –> CAC = His –> His)

Why do this kind of mutation? Might want to make a change that does alter the protein but you might want some way to detect that the change has occured. So you mutate the sequene right ext to it to make restriciton site so you can cut it and see if a mutation has occured.

Question 11

Missense

Answer 11

A mutation in the DNA that changes the amino acid sequence of the protein (CAT –> CGT = His –> Arg)

The point is to alter 1 protein by altering 1 a.a. and not effect the a.a. sequence or proteins surrounding the mutation.

Question 12

Frameshift

Answer 12

An insertion/deletion in the DNA that does not involve and multiple of 3 a.a.

The result is a change in the entire amino acid coding sequence from that point forward(GGA CAT GAG TAT GCA –>GGA CAG AGT ATG CAG = Gly His Glu Tyr Ala –> Gly Gln Ser Met Gln)

Often times it results in a stop codon

Question 13

Nonsense

Answer 13

a mutation in the DNA that generates a STOP codon (CAG –> TAG = Glu –> STOP)

TAG, TAA, TGA = STOP

Question 14

Classes of Mutagenesis Reactions

Answer 14

1. Random = mutagenesis where DNA change and the site of the change cannot be controlled . ex = PCR mutagenesis with Mn²⁺
2. Semi-Random = mutagenesi where investigator can control the site or type of mutation, not both. Ex. = Restriction site a lationn, chemical mutagenesis, gene deletion
3. Specific = site and type of mutation are controlled. Ex = Site-directed mutagenesis, SOE-PCR

Question 15

PCR mutation with Mn²⁺

Answer 15

DNA plymerase requires Mg²⁺ as a cofactor for DNA replication.

Mn²⁺ can substitue, however, the fidelity decreases dramatically.

Compination of both = investigator controls the rate of mutagenesis. It is completely random, further methods needed to determine where/what mutations have occured

Question 16

Restriction Site ablation to generate frameshift

Answer 16

EcoRI example

Cut DNA with EcoRI and produce 5’ overhangs (4 bp, AATT). You can do 1 of 2 things:

1. Remove overhang with nuclease (Mung Bean) to produce a 4 bp deletion
2. Fill in the overhang with polymerase to produce 4bp insertion.

Ligate blunt ends.

Question 17

Chemical mutagenesis

Answer 17

Use bisulfate to deaminate C and it converts to U. C pairs with G, U pairs with A. Increase the concntration of bisulfate = increase the # of C converted to U. In subsequent replications, T will now be inserted where the C –> U (Result: C-G becomes T-A)

Question 18

Gene deletion

Answer 18

Old Method: Gene of interest (GOI*) is cut with 2 restriction enzymes, more near outside than the middle. Cut peice is replaced with selectable marker (SEL⁺).

Result = GOI* has been mostly removed and replaced with SEL⁺ = null mutation (most likely)

Null mutiation = function of gene is completlly disrupted

Question 19

Site Directed mutagenesis

Answer 19

Any mutation in any location. This technique takes advantage of the fact that the DNA primer will be permenantly incorperated into the DNA. Meaning - any mutation in the primer = mutation in the DNA.

Problem = cells have ways to detect whether or not DNA has a mutation.

Question 20

Bacterial mismatch repair system (MMR)

Answer 20

Relies on methylated DNA. Dam (deoxyadenosine methylase) recognizes a specific DNA sequence in bacterial DNA and methylates the A in that sequence.

When a new strand is synthesized, there is a delay in the methylation fo the new strand.

Since the new strand is not yet methylated, the MMR system can tell the difference between the original and the new strand and it will repair the DNA to contain the sequence of the original strand.

Result:

1. both strands methylated = no repair on either strand
2. 1 strand (hemi) methylated = repair of nonmethylated strand
3. Neither strand methylated = 50% repair of mutated strand and 50% repair of original strand.

Where is the mutation in this instance?

Question 21

Site Directed by pass of MMR

Answer 21

Senerio: Mutaion occurs in vitro in abscence of DAM = not methylated mutant = How to prevent MMR?

Solution: After mutagenesis but before transformation, treat with DpnI (a restriction enzyme that requires DNA lmethylation to cleave the DNA).

Results:

1. Both methylated = cleave both
2. hemi = cleaves methylated strand
3. neither = no cleavage

*During mutagenesis, only original will be methylated and only this original will be cleaved, leaving the mutation intact.

Question 22

Splicing by Overlap Extension (SOE) PCR

Answer 22

Requirements:

1. Complementary mutagenic primers
2. Flanking primers to define the endpoints of final PCR product
3. All other necessary PCR components

Steps:

1. 1st PCR = use 1 mutagenic primer and 1 flanking primer to generate 2 PCR products
2. 2nd PCR = combine 1st PCR products with flanking primers only.

Result = PCR product containing desired mutations

Question 23

Basic uses for DNA sequencing

Answer 23

1. Identifying a gene from a genetic screen
2. Determining if a gene has a mutation
3. Confirming the creation of a sequence change after mutagenesis
4. Determinging the sequence of an entire genome

Question 24

DNA sequencing technologies

Answer 24

1. Chemical: utilizes chemical cleavage of DNA at particular bases to determine the sequence
2. Chain termination: utilizes a nucleotide triphosphate in which no further nucleotides can be added, thereby terminating DNA synthesis. Since terminating DNA is known, sequence can be determined
3. Pyrogenic: utilizes a photochemical reation that can be linked to nucleotide addition. since nucleotide is known, sequence can be determined

Question 25

Chemical DNA sequencing

Answer 25

Developed my Maxam and Gilert in the late 1970’s

Involves cleavage by chemicals at specific bases by 2-step catalytic process

1. Treat DNA with dimethylsulfate (purine specific) or hydrzine (pyrimidine specific) to remove base. Conditions can be adjusted to cleave certain bases.
2. Treat with piperidine (cleaves phosphodiester bond where base was removed). Prior to chemical treatment and cleavage the DNA must be made single-stranded and labeled. This can be done by attaching radioactive phosphate to the 5’end of the DNA. This allows DNA to be detected on the gel (G rxn only, G and A rxn, T and C rxn, C rxn only).

Run on an acrylamide gel - able to seperate by 1 bp

Question 26

Chain terminating nucleotide

Answer 26

Dideoxynucleotides have a H on the 3’ and 2’

Since additions of nucleotides by DNA polymerase requuires a 3’ OH, no further addition can be made on a ddNTP. Addition of ddNRP to a sequence results in termination of synthesis

Developed by Sanger in late 1970’s

Must use ddCTP with 1/10 normal amount of dCTP = differenct size fragments

Repeat procedure with ddATP, ddGTP, and ddTTP

Question 27

Fluorescent chain termination

Answer 27

Same as with the acrylamide gel method but instead of 4 seperate rxns, you do 1 rxn with fluorescent ddATP, ddTTP, ddCTP, ddGTP and unlabeled dATP, dTTP, dCTP, and dGTP.

Read with capilary electrophoresis (mircocapilary with gel-like material in it) uses a laser to excite fluorescent tagged ddNTP’s and a CCD camera to read emissions = Electropherogram aka Histogram

Question 28

Identifying heterozygosity by sequencing

Answer 28

Used in identifying genetic diseases

Homozygous WT chromosome produces 1 peak

Heterozygous produces 2 smaller overlapping peaks

Question 29

Sequencing the human genome

Answer 29

3 x 10⁹bp with 2 groups in the 90’s and 00’s.

1 group publicly funded (International Human Genomic Sequencing Consortium)

1 group privately funded (Celera)

Both used shotgun sequencing

Question 30

Pyrosequencing

Answer 30

1. dNTP’s added 1 at a time
2. After incorperation, pyrophosphate (PPi) is produced
3. ATP sulfurylase uses PPi to generate ATP
4. Luciferin is added in the prescence of the ATP and luciferase converts this to the oxyluciferin and light
5. The intensity of the light is detected to signal how many of that nucleotide are incorperated
6. Apyrase breaks down the extra nucleotides and ATP in the rxn mixture, process repeated
7. Developed by Nyren and Nonaghi in mid/lat 1990’s