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BIOL 112 Lecture Notes - Lecture 28: Restriction Fragment Length Polymorphism, Restriction Site, Restriction Enzyme

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Verified Note
amaranthtiger876
11 Apr 2019
School
McGill University
Department
Biology
Course
BIOL 112
Professor
Joseph Dent, Frieder Schock

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BIOL 112 Lecture Notes - Lecture 1: Radiocarbon Dating, Covalent Bond, Atomic Number

2

BIOL 112 Lecture Notes - Lecture 2: Covalent Bond, Hydrogen Bond, Chief Operating Officer

28

BIOL 112 Lecture Notes - Lecture 28: Restriction Fragment Length Polymorphism, Restriction Site, Restriction Enzyme

Document Summary

From last lecture: we saw how we can use genetic engineering in cloning to learn something about a gene that is mysterious. About a gene that is causing a disease. We know the symptoms of that disease, but we don"t know what went wrong. Because we know that the gene is a piece of dna on the chromosome somewhere, we can learn about where is might be by mapping. Since they are linearly aligned on the chromosome, we can narrow it down to get closer to the actual piece of dna that has the gene. Fruit fly phenotype is good to use for mapping (single mendelian trait) but we don"t have these in humans . So, the solution we found was the restriction fragments like polymorphism. We realized that scattered throughout the genome are these restriction sites (just occurring randomly in the.

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Related Questions

(tautomerization) The most common form of guanine is the ketoform, not the enol form.

True

False


What term describes the allowed degree of base pair matchingduring annealing, varying from lots of base-pair mismatches allowedto allowing only perfect base-pair matching.

stringency

aneuploidy

polyploidy

quantitative PCR

restriction fragment length polymorphism


I have an individual with a disease we will call SSI-itis,brought on by extreme exposure to cannabis. I do a southern andfind the gene affected. Another of his classmates exhibits theexact same symptoms, but that gene in hiw is wt. A southern in thesecond student finds a mutation in another gene. This is an exampleof

pleitropy

allelic heterogeneity

locus heterogeneity

dominant traits

recessive traits

penetrance


If a person carrying a dominant allele does not demonstrate thephenotype strongly, but still differs from wt, thisdemonstrates?

haploinsufficiency

penetrance

expressivity

monogenic traits

polygenic traits


I find three alleles for a gene in a fruit fly. One allelecreates the ability for the fly to eat graphite. Another allelecreates the ability for the fly to eat coal. The third alleleallows the fly to eat diamond. These alleles are all dominant, andwt cannot eat any of these substances. Which statement below is themost true?

Upon mating a bunch of the flies carrying these alleles, Ishould not find a fly that can eat graphite, coal, and diamond.

I would consider the ability to eat these substances an exampleof pleitropy because they are all carbon.

I would expect the ability to eat diamond to be dominant to theability to eat graphite.

These flies are demonstrating locusheterogeneity.


Expression vectors in prokaryotes do not make functionaleukaryotic gene products in bacteria very well because

the codon sequence for prokaryotes is different than the codonsequence in eukaryotes

there are no disulfide bridges formed in proteins normally madein prokaryotes

prokaryotic expression vectors cannot translate eukaryticsequences

eukaryotic genes have introns, and prokaryotes don't

eukaryotic genes have exons and prokaryotes don't


Which of the following is not used in PCR amplification ofDNA?

Double-stranded DNA template

Oligonucleotide primers

DNA polymerase

DNA ligase

ddNTPs


I create a knockout mouse using the agouti/black fur systemdiscussed in the online lecture and in the book. When I cross theagouti offspring of the originally engineered mouse, I find a ratioof 2 agouti mice to 1 black furred mouse. What is the bestexplanation?

The gene knocked out was recessive.

The gene knocked out was dominant.

The gene knocked out was a lethal gene.

The knockput was integrated into a random spot, and did notknockput the original gene.


A loss of function mutation is usually recessive, but canactually be a dominant mutation because of what effect?

haploinsufficiency

dominant negative mutations

allelic heterogeneity

locus heterogeneity

pleitropy


A genetic carrier of a disease can have a form of the diseasethat only shows intermittent symptoms, while the full homozygousrecessiuve individual would show all of the symptoms of thedisease.

True

False


Wild type, or wt, refers to the genotype that is the typicalgenotype found in nature. Now read that again. Once more. Nowanswer.

Answer

True

False



2.Conclusion:

What have you learned from your study?

Interpret your data/ results:

Do your results support of disprove your hypothesis?

Compare and contrast your result with those of previous research.

Significance of the results: “Big Picture”

What is the global impact of your results?

What questions remain unanswered?

What kinds of experiments can come out of your study?

i have lab for biology and i need help with the Lab Report

the lab name is Bacterial Transformation Lab

1. Introduction:

Introduce the background and the nature of the problem your experiment is investigating.

Summarize the current knowledge of your subject.

Use examples of other research that have examined the same or a similar topic as your experiments.

Ending your introduction:

State your hypothesis

State your predictions

Give a brief statement about how you tested your hypothesis.

please help me with it is important for my lab class tomorrow

that is the lab i put the info please help me for the intro and for number 2 please be clear please

i would be happy if you help me it is due tommorrow i realy need your help on the intro and number 2 please be clear and show clear work hope you do it because it is due tomorrow

Bacterial Transformation Lab

Genetic engineering is one of the newest applications of basic knowledge in biology. It involves the isolation of specific genes from one organism and the insertion of those genes into a second organism of the same, or even different, species. Remember that a gene is a piece of DNA which codes for a product, usually a protein, which gives an organism a particular trait. The development of these techniques has generated considerable excitement and answered many questions, but it has also raised questions. Many scientists and others see genetic engineering as offering opportunities to make life better, for example, by exploring how genes are regulated, by curing diseases of genetic origin, by adding desirable traits to agricultural crops such as the ability to fix nitrogen in corn and pest resistance in cotton, and by genetically altering bacteria with genes enabling them to digest oil spills.

For almost 100 years, it has been possible to isolate DNA from cells, but only in the last 40 years or so has its function been understood. Your textbook discusses the work of such investigators as Fred Griffith and Oswald Avery, who demonstrated that if DNA was isolated from a pneumonia-causing strain of bacteria and added to cultures of nonvirulent bacteria, the recipient cells were transformed into virulent types that caused pneumonia. These transformation experiments were the first to show that genes could be artificially transferred in the laboratory. Since the time of those experiments (Avery's work was done in the 1940s), our knowledge of gene structure, function and control has increased astronomically. The application of this knowledge has created the field of genetic engineering.

Modern genetic engineering techniques depend on three critical factors:

A suitable host organism in which to insert the foreign gene;

A vector to carry foreign genes into the host; and

A means of isolating the host cells that have taken up the foreign gene.

The host organism

The most commonly used host organism is the gut bacterium Escherichia coli (E. coli). Its chromosome is a single large circular DNA molecule, called the genomic DNA, containing about 4 x 106 base pairs. Not every cell will take up foreign DNA when exposed to it, but E. coli grows rapidly, dividing in less than 20 minutes, so that a single cell can give rise to a billion genetically identical descendants in a matter of 10 hours. Thus, a few transformed cells can provide a large number of offspring within a day. The strain we are using is K-12.

The vector

A vector is a DNA molecule used as a vehicle to carry genes from one organism to another. This DNA molecule can be modified by gene-splicing techniques using restriction enzymes and ligases so that it carries the genes of interest (essentially a cut-and-paste operation). Sometimes viruses are used as vectors, but for E. coli, plasmids are often used. A plasmid is a small circular DNA molecule containing 1000 to 200,000 base pairs. Plasmids exist naturally in many strains of E. coli and other bacteria. Plasmids replicate as the cell grows, independent of replication of the genomic DNA. These plasmids are transmitted to each of the product cells during cell division. Plasmids carrying genetic information that is beneficial to the host cell are maintained in a given population by natural selection. Plasmids frequently carry genes that confer antibiotic resistance, an obvious benefit.

When E. coli cells are exposed to plasmids or any other foreign DNA, some of the cells, called competent cells, will take up the DNA. We can induce cells to become competent by treating them with divalent cations, such as Ca++, followed by a brief exposure to high temperature, called a heat shock treatment. Exactly how this treatment causes cells to become competent is not understood, but it is described as making the membranes "leaky" so that large DNA molecules (like plasmids), which would not normally pass through the cell membrane, can enter the cell. This treatment does not make all cells competent, but it greatly increases the number of competent cells. Once in a cell, the plasmids replicate and are passed to the product cells during cell division.

The selective medium

The last requirement for genetic engineering is a means of isolating host cells that have taken up the gene of interest. The techniques used depend on the growth requirements of the host organism. For example, each species of bacteria has particular nutrient requirements. The composition of the growth medium (agar or broth) can be modified to allow growth of only certain bacteria -- for example, by adding or removing nutrients or other chemicals from the medium mixture. This modified medium is called a selective medium, because it selects for the growth of only some species. By introducing an antibiotic such as ampicillin into the medium in which the bacteria are grown, you will select for growth of only those bacteria carrying the antibiotic-resistance gene on newly acquired plasmids. In a matter of days, billions of antibiotic-resistant cells, all with the plasmid, can be grown.

Genetic engineers use a modification of this technique to insert a foreign gene into E. coli. In simplified terms, the plasmid (already carrying an ampicillin resistance gene) is modified by inserting the gene of interest. The gene of interest has been isolated from another source; for example, the gene for human insulin has been isolated from the human genome. The modified plasmid, carrying the inserted gene, is then mixed with competent E. coli and the bacteria are placed in a medium containing ampicillin. Only those cells that have the plasmid will grow in the medium, and they are also the cells carrying the insulin gene (in their plasmids). These selected cells can now be used to manufacture insulin, which can be used for insulin therapy by diabetics.

The genes

In the following experiment, you will insert a plasmid into E. coli. The plasmid you will use carries the gene bla, which codes for a protein (beta-lactamase) that is secreted by the bacteria, inactivating the ampicillin in the agar and allowing for bacterial growth. The plasmid also carries an indicator gene. The indicator gene we are using was isolated from a bioluminescent jellyfish, Aequorea victoria. It codes for Green Fluorescent Protein (GFP).

The GFP gene has been modified to include a regulator system that can be used to control the expression of the gene. Gene expression in all organisms is carefully regulated to allow for adaptation to differing conditions and to prevent wasteful overproduction of unneeded proteins. The genes involved in the breakdown of different food sources are good examples of highly regulated genes. For example, the sugar arabinose is both an energy source and a carbon source for bacteria. The bacterial genes that make digestive enzymes to break down arabinose for food are not expressed when arabinose is not in the environment. But, when arabinose is present, these genes are turned on. When the arabinose runs out, the genes are turned off.

Arabinose initiates transcription of these genes by promoting the binding of RNA polymerase. In the genetically engineered pGLO DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene GFP. When bacteria with this plasmid are grown in the presence of arabinose, the GFP gene is turned on and the bacteria glow green in UV light. When arabinose is absent form the growth medium, the GFP gene remains turned off and the colonies appear white. This is an example of the central molecular framework of biology in action: DNA ? RNA ? protein ? trait.

In summary, you will render E. coli cells competent to take up plasmid DNA. The plasmid carries two identifiable phenotypic markers:

The beta-lactamase or bla gene codes for resistance to the antibiotic ampicillin. We will use this to select for bacteria that have taken up the plasmid.

The pGLO gene codes for Green Fluorescent Protein (GFP), which is switched “ON” if arabinose is present.

Safety Procedure

For your own safety and the safety of others, it is essential that you follow all instructions about handling bacteria and disposal of materials. When you finish work for the day, swab your table with disinfectant. WASH YOUR HANDS thoroughly with soap before leaving the lab for any reason, and at the end of the day after you have cleaned your table. Report any spills to your instructor.

List of Materials

Prepared agar plates (2 LB, 2 LB/amp, 1 LB/amp/ara)

Transformation solution (50 mM CaCl2, pH 7.4)

LB broth

1 pack of inoculation loops

Sterile transfer pipets

1 foam microtube holder w/ tubes

E. coli starter plate

1 red biohazard bag for loop disposal (common use – Loops/pipets only - NO PAPER!!!)

Expression vectors inprokaryotes do not make functional eukaryotic gene products inbacteria very well because

Answer Not sure which one ?!

· the codon sequence for prokaryotes is differentthan the codon sequence in eukaryotes

· there are no disulfide bridges formed in proteinsnormally made in prokaryotes

· prokaryotic expression vectors cannot translateeukarytic sequences

· eukaryotic genes have introns, and prokaryotesdon't

· eukaryotic genes have exons and prokaryotesdon't


I create a knockout mouseusing the agouti/black fur system \. When I cross the agoutioffspring of the originally engineered mouse, I find a ratio of 2agouti mice to 1 black furred mouse. What is the bestexplanation?

Answer not sure which one?!

· The gene knocked out was recessive.

· The gene knocked out was dominant.

· The gene knocked out was a lethalgene.

· The knockput was integrated into a random spot,and did not knockput the original gene.

A restriction enzyme cuts DNAand leaves the following end

xxCTGCA
xxG

Which of the following could be the sequence of the correspondingend of the other end of the cut DNA?

Answer

· xxG
xxCTGCA

· xxC
xxGTGCA

· xxCCGAT
xxG

· xxGGCTA
xxC


An SNP always occurs dueto

Answer

· a one base-pair change.

· a three base-pair change.

· a single restriction site change.

· a single amino acid change.

· None of these.


I PCR out a mutated gene in apatient with Lisenbee chorea (the inability to dance in acoordinated fashion) and I compare it to another patient with thatsame disease phenotype. One subject had a mutation on chromosome 4,and the other subject couldn't rock it because of a mutation onchromosome 5. This is an exampe of

Answer

· pleitropy

· locus heterogeneity

· allelic heterogeneity

· dominant negative mutation

· penetrance

In his experiments, Mendelnoted that when two traits are involved in a genetic cross, theyare inherited independently of each other. Though Mendel didn'tknow about chromosomes, this still holds true (mostly)because

Answer

· genes on the same chromosome are often mixedthrough meiotic crossover during the formation ofgametes.

· variant alleles of the same gene do not interferewith the phenotypic expression of the gentoype in different geneticloci.

· most higher organisms are diploid, making only twoalleles available for every genetic locus, and there is a 50/50chance of geting either allele from a single parent.

· expression of phenotypes can vary from thegenotypes because of encironmantal influences.

· chromosomes often recombine

Anticipation is caused by amutation that increases in expressivity over subsequentgenerations.

Answer

· True

· False

Question 44

I have a genotype that shouldproduce a specific phenotype, but some of the individuals with thegenotype do not demonstrate any evidence of the phenotype. I wouldconsider this an example of

Answer

· allelic heterogeneity.

· locus heterogeneity.

· penetrance.

· expressitivity.

· sheer luck.

Question

Which of the following doesnot occur during the PCR reactions?

Answer

· Changes in the reaction temperature by athermocycler

· Denaturation at high temperatures

· Synthesis of oligonucleotide primers

· Extension of primers by DNA polymerase

· Annealing of oligonucleotide primers

Question

DNA markers, or variantnon-coding regions of DNA, are DNA polymorphisms that are usefulfor genetic mapping.

Answer

· True

· False

A mosaic is an organismwith

Answer

· multiple genotypes within one organism

· multiple alleles within one genotype

· more than one color of fur

· transgenes added to the zygote beforedevelopement

· a wt phenotype but a mutated genotype

Question

Genotype causesphenotype.

Answer

· No, gentoype just influences phenotype.

· Yes, genotype is the DNA sequence that createsphenotype.

Question

A true genetic chimera can becreated by

Answer

· mutating a gene early on in the development of anorganism resulting in different alleles being present in theadult

· multiple fertilized eggs or zygotes fusing to formone embryo

· adding a transgene to the genome of an organismduring fetal development only

· adding cells of a different species to an adultorganism

· adding a transgene to the genome of an animal atany stage in development

Question

The ABO blood group can bestbe explained by the concept of _______.

Answer

· dominant traits

· recessive traits

· allelic heterogeneity

· locus heterogeneity

· vampirism

If a loss of functionmutation creates a dominant phenotype, it may be becauseof

Answer

· haploinsufficiency

· penetrance

· expressivity

· allelic heterogeneity

· locus heterogeneity

Please select the best matchfor each.

Answer

· PCR: Technique used to generatecopies of a DNA fragment

· Southern blot : Screeningprocedure used to assay DNA fragments

· RFLPs : DNA fragments generatedby restriction enzymes that vary in length betweenindividuals

· Northern blot : Screeningprocedure used to assay mRNA fragments

· Electrophoresis Movement ofmacromolecules in an electrical field

Answer

A.

Screening procedure used toassay DNA fragments

B.

Movement ofmacromolecules in an electrical field

C.

Screening procedure used toassay mRNA fragments

D.

DNA fragments generated byrestriction enzymes that vary in length betweenindividuals

E.

Technique used to generatecopies of a DNA fragment

A gene mutates, and theprotein produced has a novel way of interacting with the cell, andcreates a new phenotype because of this new functionality. This iscalled

Answer

· pleitropy

· locus heterogeneity

· allelic heterogeneity

· dominant negative mutation

· gain of function dominant mutation

Question 62

Mutations in the somaticchromosomes may be inherited by the next generation.

Answer

· True

· False


A degenerate PCR primer withmany variant sequences must be used to make multiple copies of agene

Answer

· if only the protein sequence of the gene productis known to construct the primers

· if the DNA you are using is cDNA to constructthe primers

· if the DNA you are using is genomic DNA to becopied

· if the DNA you are trying to copy iscDNA

· if the vector is prokaryotic and the transformedcell is eukarytotic

Question

Please select the best matchfor each term.

Answer

· Restriction enzyme


· PCR C

· Plasmid

· YAC A

· Dideoxynucleotide

Answer

A.

Endonuclease isolated frombacteria

B.

Eukaryotic cloningvector

C.

Method for amplification ofDNA sequences

D.

Small circular fragment ofDNA

E.

Molecule used in DNAsequencing

Question

A couple goes on MauryPovitch, and the results are in: you are not the father. But noother man impregnated the female (granted, unlikely for a MauryPovitch guest, but work with me here) and he must be the father.DNA analysis claims otherwise, though the child definitely wasmom's (poor thing). What may be going on here?

Answer

· the child is parthenogenic because the motheractually impregnated herself like a shark, and the child's DNA isall mom's

· the child had a mutation that changed the genethat is used to trace paternity

· the child is a mosaic because he is actually a setof twins fused early during fetal development, and therefore camefrom 2 eggs and 2 sperm

· the dad may have germ-line mosaicism, meaning thatthe genotype of his sperm is different from his somaticgenotype

· mitochondrial DNA only comes from mom, so there isno way to tell whobthe baby's father is