module 9 Flashcards
What are restriction endonucleases?
Enzymes that cut DNA at specific sequences, often used in genetic research and biotechnology.
What is the role of ligases in molecular biology?
Ligases are enzymes that glue or join DNA fragments together by catalyzing the formation of phosphodiester bonds.
Which enzymes are responsible for synthesizing DNA and RNA?
Polymerases are the enzymes that synthesize DNA (DNA polymerase) and RNA (RNA polymerase).
What is the function of polymerases in DNA and RNA synthesis?
Polymerases add nucleotides to a growing DNA or RNA strand, using a template strand to guide the sequence.
What is reverse transcriptase and how does it differ from other polymerases?
An enzyme that makes DNA from RNA.
How do restriction endonucleases cut DNA?
They recognize specific short DNA sequences (often palindromes) and cut both strands of DNA at these sites.
Can reverse transcriptase be used to synthesize RNA from a DNA template? Why or why not?
No, it makes DNA from RNA.
How are restriction endonucleases and ligases used together?
Endonucleases cut DNA, and ligases join it into vectors during cloning.
What is a restriction digest?
A process where restriction enzymes cut DNA into smaller fragments.
What is electrophoresis used for?
Separates DNA fragments by size using an electric field.
What is molecular cloning?
A technique to insert a gene into a plasmid vector and introduce it into a host cell.
What is PCR (Polymerase Chain Reaction)
A method to amplify specific DNA sequences rapidly.
What is DNA sequencing?
Determining the exact order of nucleotides in a DNA molecule.
What is hybridization in molecular biology?
The process of binding complementary DNA or RNA strands together.
What are microarrays used for?
Analyzing gene expression by detecting hybridization of labeled DNA/RNA to a grid of probes.
What is the natural source of restriction endonucleases?
They are naturally found in bacteria, where they protect against viral DNA.
What is the main function of restriction endonucleases?
They cut double-stranded DNA without damaging the nitrogenous bases.
How does the length of a restriction enzyme’s recognition sequence affect fragment size
Longer recognition sequences produce longer DNA fragments.
Is restriction digestion time-dependent?
Yes, the efficiency of digestion can depend on the incubation time.
Why isn’t bacterial DNA digested by its own restriction enzymes?
Bacterial DNA is protected by methylation, which prevents recognition by the enzymes
whats a sticky 5 prime end
results from staggered cutes leaving single stranded dna. These sticky ends can easily pair with complementary sticky ends from other DNA fragments, making them useful for molecular cloning and recombination
whats ecorI and what does it do
it is a restriction enzyme known as Escherichia coli.EcoRI recognizes and cuts the DNA sequence GAATTC. it does a sticky cut.
sticky cuts are _____
staggered
whta is HindIII and what does it cut.
HindIII recognizes the palindromic sequence AAGCTT,. it is a restriction enzyme called haemophilus influenzae
What does electrophoresis do?
Separates molecules using a matrix and an electric field.
How are fragments separated in electrophoresis?
By size and charge of the molecule.
Why is DNA negatively charged?
Each phosphate in the sugar-phosphate backbone carries a negative charge.
How does DNA size affect its movement in electrophoresis?
Smaller DNA fragments travel faster than larger ones in the electric field.
electrophoresis process
Electrophoresis is a technique used to separate molecules like DNA or proteins by size and charge. A gel (usually agarose for DNA) is prepared, and samples are loaded into wells at one end. When an electric current is applied, negatively charged DNA moves toward the positive electrode. Smaller fragments travel faster, while larger fragments move slower, separating based on size. After running the electric current for a set time, the gel is stained (e.g., with ethidium bromide) and viewed under UV light. The DNA appears as bands, and their size is compared to a molecular weight marker. This method is widely used in genetic analysis and protein separation.
Why do we isolate and amplify DNA fragments?
To analyze and use specific regions of DNA.
What is molecular cloning?
The process of inserting a piece of DNA into a vector and then transferring it into a bacterium.
How does molecular cloning work?
The vector carrying the DNA is placed in a bacterium, which replicates and makes multiple copies of the DNA as it grows.
How are DNA copies harvested after cloning?
The DNA is extracted from the bacterium after it has replicated and grown.
How can we amplify DNA without cloning it in bacteria?
By using Polymerase Chain Reaction (PCR) to amplify the DNA if part of the sequence is known.
What are plasmids used for in molecular cloning?
Plasmids are small circular DNA molecules used as vectors to carry foreign DNA.
How are plasmids prepared for cloning?
Plasmids are cut open with restriction enzymes to create sticky ends for ligation.
How is human DNA used in molecular cloning?
Human DNA is cut into smaller pieces using restriction enzymes.
What happens during the ligation step?
Human DNA fragments are ligated (joined) into the open plasmid using DNA ligase.
What is transformation in molecular cloning?
Transformation is the process of introducing the recombinant plasmid back into bacteria.
What is a recombinant plasmid?
A recombinant plasmid is a plasmid carrying foreign DNA (e.g., human DNA).
How is successful plasmid introduction selected for in bacteria?
By using an ampicillin resistance gene in the plasmid.
What is the role of the ampicillin resistance gene in molecular cloning?
It allows for the selection of bacteria that have incorporated the plasmid by making them resistant to ampicillin.
Why do we use ampicillin for selection?
Only bacteria with the recombinant plasmid, which carries the ampicillin resistance gene, can survive and grow on media containing ampicillin.
What does the lac operon do in molecular cloning?
The lac operon contains the lacZ gene, which codes for beta-galactosidase that breaks down lactose into glucose and galactose.
What is the function of beta-galactosidase in cloning?
Beta-galactosidase cleaves lactose into glucose and galactose, and in the presence of X-gal, it produces a blue color.
How is the lac operon used to select for successful DNA insertion?
If the lacZ gene is intact and functional, beta-galactosidase cleaves X-gal and turns the colony blue. If the lacZ gene is disrupted by inserted foreign DNA, the colony stays white.
What happens if the lacZ gene is functional after ligation?
The colonies turn blue because the beta-galactosidase enzyme cleaves X-gal and produces a blue product.
What color are colonies with successful DNA insertion (disrupted lacZ)?
Colonies with successful DNA insertion are white, indicating the lacZ gene has been disrupted by the foreign DNA.
What happens if foreign DNA is inserted into a plasmid at the lacZ gene?
The lacZ gene is disrupted, preventing the production of beta-galactosidase.
Why do colonies with disrupted lacZ gene remain white?
hey cannot cleave X-gal, so no blue color is produced.
What color are the colonies that carry the foreign DNA?
The colonies are white, indicating that the foreign DNA has successfully disrupted the lacZ gene.
How can you identify bacteria that have successfully taken up foreign DNA?
Look for white colonies, as they indicate successful insertion of foreign DNA into the plasmid.
What is an example of using molecular cloning to produce a human protein?
Human insulin is a common example, produced by inserting the human insulin gene into bacteria.
How is the human insulin gene isolated for cloning?
The insulin gene is extracted from human DNA and cut into smaller pieces using restriction enzymes.
How is the insulin gene inserted into a plasmid?
The plasmid is cut open with restriction enzymes, and the insulin gene is ligated into the plasmid using DNA ligase.
What is a recombinant plasmid?
A recombinant plasmid is a plasmid that carries foreign DNA, such as the human insulin gene.
How is the recombinant plasmid introduced into bacteria?
The recombinant plasmid is introduced into bacterial cells (typically E. coli) through transformation.
What happens after bacteria take up the recombinant plasmid?
The bacteria replicate the plasmid and produce human insulin as the insulin gene is expressed in the bacterial cells.
Why is E. coli used in the production of human insulin?
E. coli can quickly replicate foreign DNA and produce the proteins encoded by that DNA, making it efficient for producing large quantities of human insulin.
What is the role of restriction enzymes in the cloning of human insulin?
Restriction enzymes cut both the plasmid and the insulin gene into fragments with sticky ends, allowing for the ligating of the gene into the plasmid.
How does ligase help in the molecular cloning process?
DNA ligase joins the insulin gene to the cut plasmid, creating a recombinant plasmid.
Who invented the Polymerase Chain Reaction (PCR)?
Kary Mullis and others invented PCR in the early 1980s, and he won the Nobel Prize in 1993 for it.
What does PCR mimic?
PCR mimics the cellular process of DNA replication.
What are the basic supplies needed for PCR?
The supplies needed for PCR include:
A DNA template
Primers (short pieces of single-stranded DNA)
Nucleotides (the building blocks of DNA)
DNA polymerase (an enzyme that synthesizes DNA)
What is the role of primers in PCR?
Primers are short pieces of single-stranded DNA that bind to the DNA template to initiate the replication process.
What is the function of DNA polymerase in PCR?
DNA polymerase synthesizes new strands of DNA by adding nucleotides to the primers.
Why are nucleotides important in PCR?
Nucleotides are the building blocks that the DNA polymerase uses to create new DNA strands.
What is the DNA template used for in PCR?
The DNA template provides the sequence that will be copied and amplified during the PCR process.
What is the DNA template in PCR?
The DNA template provides the sequence to be amplified and copied during PCR.
What is the DNA template in PCR?
The DNA to be amplified.
What type of primers are used in PCR?
DNA primers.
Are both strands leading in PCR?
yes, both strands are leading in PCR.
How many DNA polymerases are involved in PCR?
One DNA polymerase (with less efficient proofreading).
What is required for the PCR reaction to occur?
Free nucleotides and varying temperatures.
What is the DNA template in cellular DNA replication?
The DNA to be replicated.
What type of primers are used in cellular DNA replication?
RNA primers.
What types of strands are involved in cellular DNA replication?
Leading and lagging strands.
How many DNA polymerases are involved in cellular DNA replication?
Several DNA polymerases.
What enzymes are required for cellular DNA replication?
A host of enzymes, including helicase, topoisomerase, and DNA polymerase.
What is the role of the left primer in PCR?
The left primer binds to the 3’ end of the template strand, initiating DNA synthesis on the leading strand.
What is the role of the right primer in PCR?
The right primer binds to the 3’ end of the complementary strand, initiating DNA synthesis on the lagging strand.
What is the target region in PCR?
The target region is the specific DNA sequence between the left and right primers that will be amplified.
How is mRNA isolated for making cDNA?
mRNA is isolated from cells.
What does isolating mRNA allow researchers to study?
It allows researchers to study which genes were expressed in the tissue at the time of isolation.
Why do we convert mRNA to DNA?
To understand which genes were expressed by making cDNA.
What is a cDNA library?
A cDNA library is a collection of complementary DNA (cDNA) molecules synthesized from mRNA.
What is the advantage of using mRNA to create a cDNA library?
It represents all the genes expressed in the tissue at the time of isolation, enabling researchers to study gene expression.
What enzyme is used to convert RNA into DNA?
Reverse transcriptase.
What type of enzyme is reverse transcriptase?
It is a retroviral enzyme.
What is the function of reverse transcriptase?
Reverse transcriptase converts RNA into complementary DNA (cDNA).
When was reverse transcriptase discovered?
In 1970.
Why is reverse transcriptase important in molecular biology?
It allows the synthesis of DNA from RNA, enabling the creation of cDNA libraries and the study of gene expression.
What is the first step in reverse transcription?
use an oligo(dT) primer to fish around with polyT primer. various nucleotides used by complementary fashion using reverse trnaciptase. trying to make DNA so use TTP. end up with CDNA and mRNA.
who discovered retrovirus
david baltimore. and dr temmin
What is the second step in reverse transcription?
CDNA will fold back on itself because messenger RNA was digested and nucleotides were added again. and second complementary strand of CDNa will be made. at end we have CDNA double helix.
libraries are
colelction of clones in a vector.
what are examples of libraries
genomic library, CDNA library
What is the purpose of a DNA library?
o store and preserve a large number of DNA fragments for research, such as gene sequencing, cloning, or functional analysis.
What is a genomic library?
A genomic library is a collection of DNA fragments that represent the entire genome of an organism, including both coding and non-coding regions.
What is a cDNA library?
A cDNA library is a collection of complementary DNA (cDNA) clones synthesized from mRNA, representing only the expressed genes (coding regions) of an organism at a specific time.
How is a genomic library created?
Genomic DNA is fragmented and inserted into vectors (such as plasmids or bacteriophages), then cloned into host cells for storage.
How is a cDNA library created?
mRNA is isolated, reverse transcribed into cDNA, and then inserted into vectors for cloning in host cells.
What is the main difference between a genomic library and a cDNA library?
A genomic library includes all genomic sequences (both coding and non-coding regions), while a cDNA library contains only the coding sequences (expressed genes) transcribed from mRNA.
Why would a researcher use a cDNA library instead of a genomic library?
A cDNA library allows the study of only the actively expressed genes at a particular time, which is useful for gene expression studies.
What does a double-stranded cDNA represent in a cDNA library?
The double-stranded cDNA is a copy of the mRNA that was prevalent in the cell at the time of extraction.
How are cDNAs cloned for use in a library?
cDNAs are inserted into plasmid vectors.
What happens after cDNA is inserted into plasmids?
The plasmids are introduced into bacteria, typically through a process like transformation.
How are cDNA libraries created from bacteria?
Bacteria carrying plasmids with cDNA are cultured and stored, effectively creating a cDNA library.
Why are bacteria used to store cDNA libraries?
Bacteria can replicate the plasmids containing cDNA, allowing for long-term storage and amplification of cDNA clones.
