You want to make a recombinant DNA in which aPCR product amplified from the human genome is inserted into a plasmid vector. The polylinker of thisvector includes recognition sites for the enzymesEcoRI (5′ G^AATTC 3′) and BamHI (5′ G^GATCC3′). (The ^ symbolizes the cut site in the DNA.) PCRprimers that could amplify the fragment of humanDNA are: 5′ GCTACTTCGCGTATTCCA 3′ and5′ CCCAAGTCCTAGCCGATA 3′.a. Describe in detail how these primers would need tobe modified to create a fragment of the human genome flanked by EcoRI sticky ends so that thisfragment could be cloned easily into the plasmidvector. You will need to consider the fact that mostrestriction enzymes, including EcoRI, cannot cutDNA if the restriction site is directly at the end ofthe DNA molecule; the restriction enzyme recognition site must be at least six base pairs distant fromthe end.b. Describe a potential feature of the PCR-amplifiedregion of the human genome that could prevent youfrom using the strategy you described in part (a).c. Now describe how the primers must be modifiedto create a human DNA fragment with an EcoRIcompatible single-stranded overhang at one endand a BamHI-compatible overhang at the otherend. (Two possibilities exist; you need todescribe only one. Assume that a BamHI site alsomust be at least six base pairs from the end ofthe DNA.) Why might you want to make sucha fragment?

Question

You want to make a recombinant DNA in which a
PCR product amplified from the human genome is inserted into a plasmid vector. The polylinker of this
vector includes recognition sites for the enzymes
EcoRI (5′ G^AATTC 3′) and BamHI (5′ G^GATCC
3′). (The ^ symbolizes the cut site in the DNA.) PCR
primers that could amplify the fragment of human
DNA are: 5′ GCTACTTCGCGTATTCCA 3′ and
5′ CCCAAGTCCTAGCCGATA 3′.
a. Describe in detail how these primers would need to
be modified to create a fragment of the human genome flanked by EcoRI sticky ends so that this
fragment could be cloned easily into the plasmid
vector. You will need to consider the fact that most
restriction enzymes, including EcoRI, cannot cut
DNA if the restriction site is directly at the end of
the DNA molecule; the restriction enzyme recognition site must be at least six base pairs distant from
the end.
b. Describe a potential feature of the PCR-amplified
region of the human genome that could prevent you
from using the strategy you described in part (a).
c. Now describe how the primers must be modified
to create a human DNA fragment with an EcoRIcompatible single-stranded overhang at one end
and a BamHI-compatible overhang at the other
end. (Two possibilities exist; you need to
describe only one. Assume that a BamHI site also
must be at least six base pairs from the end of
the DNA.) Why might you want to make such
a fragment?

Expert Answer

Want to see the step-by-step answer?

Check out a sample Q&A here.

Want to see this answer and more?

Experts are waiting 24/7 to provide step-by-step solutions in as fast as 30 minutes!*

*Response times may vary by subject and question complexity. Median response time is 34 minutes for paid subscribers and may be longer for promotional offers.
Tagged in
Science
Biology

Related Biology Q&A

Find answers to questions asked by students like you.

Q: Describe nucleosome supercoiling and its relationshipto the radial loop–scaffold model of chromatinp...

A: The eukaryotic cell has a nucleus and nuclear membrane which contain genetic material wherein prokar...

Q: Describe the conditions of nutrition, pH, temperature, oxygen, andlife cycle that made it possible f...

A: Microorganisms are isolated and cultured for various reasons. The role of media in the culture, iden...

Q: Recently, scientists constructed a transgene that expresses a mutant form of Drosophila histone H3 i...

A: Histones are the alkaline proteins present in the nucleus of the eukaryotic cells. They remain bound...

Q: If a solution has a concentration of 10^-7 OH ions, how many H ions does it have?

A: pH is chemical scale that specifies the acid or basic strength of a solution. Pure water has a pH of...

Q: Differentiate between contamination, colonization, infection, anddisease, and explain some possible ...

A: The terms contamination, colonization, infection and disease are related to the interaction of an or...

Q: Define mutation. Understand classification of major types of mutations.

A: A gene is the physical and functional unit of heredity. They pass information from one generation to...

Q: Eukaryotic genomes are replete with repetitive sequences that make genome assembly from sequenceread...

A: Tandem repeats of the dinucleotide sequence are the repetitive sequences that are found at many loca...

Q: Which of these characteristics is not a major factor in adaptive oracquired immunity?a. specificity ...

A: Characteristic Specific immunity developed by lymphocytes in response to antigen termed as adaptive ...

Q: Using figure1, label the lines and arrows, and explain what ishappening at numbers 1, 2, and 3

A: The given figure shows the process of B-cell activation, antigen processing, antigen presentation, a...

Q: Discuss the factors involved in allergies and hypersensitivities.

A: Hypersensitivity Reactions: Hypersensitivity or allergy can be defined as excessive, undesirable rea...

Q: Consider a partial restriction digestion, in which genomic DNA is exposed to a small, limiting amoun...

A: The process of cutting or cleaving DNA by using restriction enzymes is called digestion or restricti...

Q: a. What is the purpose of molecular cloning?b. What purpose do selectable markers serve in vectors?c...

A: Plasmids are small, circular extrachromosomal DNA present in a cell. These extrachromosomal molecule...

Q: why do you rarely see sexual reproduction of the zygomycota on bread?

A: Zygomycota comes under the fungal kingdom. In Zygomycota, the reproduction occurs either asexually o...

Q: What is radiation? Differentiate between ionizing and non-ionizing radiation in their mode of action

A: There are two types of radiation that are majorly used. They are known as ionizing radiation and non...

Q: Due to dosage compensation by X inactivation, females can exhibit a mosaic pattern of sweat glands o...

A: X-inactivation refers to the inactivation of one of the X chromosomes that is present in pairs in ev...

Q: #6 on the first page   #7 on the second page

A: True breeding is a process of mating in which the parents would produce offspring that would carry t...

Q: 19. Which statement best describes at the molecular level why AB is the universal acceptor? a. It is...

A: Carbohydrates play a major role in cell-cell recognition. Their structural variation allows specific...

Q: Researchers know that Fru-M controls male sexualbehavior in Drosophila because inappropriate Fru-Mex...

A: In Drosophila melanogaster, sex is determined by cell and tissue-specific signals and not by hormone...

Q: Four human babies were accidentally mixed up in the maternity ward. The blood types of the babies we...

A: The blood group of a person will be decided by three genes; A, B, and O. The A and B genes are co-do...

Q: Could other inmates develop botulism from being in contact with theill prisoners?a. Yes, it can be s...

A: Botulism is a rare serious illness caused by the organism Clostridium botulinum. This bacteria produ...

Q: What is the role of the core histones in compaction ascompared to the role of histone H1?

A: DNA is a double helix structure which contains the genetic material necessary for inheritance of cha...

Q: Read “Safe at the Plate”.  List 4 different bugs, their signs & symptoms and what to do if they ...

A: Bugs are the major known agents that cause "foodborne diseases". They are pathogens such as a bacter...

Q: Define: genome, gene (structural & regulatory), chromosome, histone protein.

A: The hereditary material that is found in the cells of all living forms is referred to as genetic mat...

Q: How would the artificial mRNA5′. . . GUGUGUGU . . . 3′be read according to each of the following mod...

A: In molecular biology, a reading frame refers to a method of separating the nucleotides sequence pres...

Q: The sequence of a segment of mRNA, beginning withthe initiation codon, is given here, along with the...

A: A mutation is a change that occurs in our DNA sequence, either due to mistakes when the DNA is copie...

Q: Explain the relevance of a species RefSeq tobioinformatic studies

A: RefSeq (species reference sequence) is an open access, annotated and complete genome sequence of a s...

Q: Where does NADH originate, and what is its fate in an aerobe?

A: The full form of NADH is nicotinamide adenine dinucleotide. It takes part in various redox reactions...

Q: Unlike life cycles found in many land plants and protist's life, animals do not go through alternati...

A: Life cycle of plant starts with development of a seed and every seed has a miniature plant known as ...

Q: . The double-stranded circular DNA molecule thatforms the genome of the SV40 tumor virus can be dena...

A: Transcription is the process of synthesizing RNA (ribonucleic acid) molecule by using a template DNA...

Q: Explain what is happening during each stage of infection. Compare and contrast: systemic, local, and...

A: The progression of an infection or disease occurs in four stages and there are several patterns in w...

Q: Compare and contrast the actions of DNA polymerase and RNApolymerase.

A: Deoxy ribonucleic acid (DNA) is the genetic material of most organisms that contain coded genetic in...

Q: Would you characterize the pattern of inheritance ofanonymous DNA polymorphisms as recessive, domina...

A: The Human Genome Project was completed in 2003, and the DNA sequence of the complete human genome wa...

Q: The Ames test uses the reversion rate (His− to His+)to test compounds for mutagenicity.a. Is it poss...

A: Ames test screens for chemicals that induce mutations in bacterial cells. This test examines if a pa...

Q: Discuss mutations outside the coding sequence thatcould affect gene expression.

A: DNA is a polymer of nucleotides arranged in unique sequence in different organisms. It forms the gen...

Q: Relate tRNA’s structure to its function

A: tRNA refers to transfer RNA is a small molecule which contain 74 to 95 ribonucleotides. They are com...

Q: Discuss why it is advantageous for humans to producedifferent hemoglobins at different stages ofdeve...

A: Hemoglobin is a type of a protein present in the red blood cells of vertebrates and some invertebrat...

Q: The question is : Examples of polysaccharides that have 1,6 bonding are ___ and ____

A: Polysaccharide as the name suggests that “poly” means many and “saccharides” mean carbohydrates or s...

Q: In corn snakes, the wild-type color is brown. Oneautosomal recessive mutation causes the snake to be...

A: In the given question the wild-type color is brown. One autosomal recessive mutation caused the snak...

Q: The mitotic cell divisions in the early embryo ofD. melanogaster occur very rapidly (every eightminu...

A: Cell division is a natural process in which a parent cell divides into two daughter cells. Cell cycl...

Q: Certain individuals with mild forms ofβ-thalassemia produce, in addition to normal adulthemoglobin w...

A: Haemoglobin lepore is defined as a small group consisting of structurally abnormal haemoglobins whic...