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Terms in this set (102)
MB: What makes DNA cloning possible?
The ability of restriction enzymes to cut DNA at specific sites
MB: Cloning a gene requires...
...an enzyme that has restriction sites on both sides of that gene but not within the gene
MB: What make it possible for the fragments to combine with the DNA of a cloning vector, such as a plasmid
MB: To screen a library of bacterial colonies for clones that carry a specific gene...
...a relatively short, single-stranded nucleic acid probe is hybridized to the DNA of that gene
MB: (TRUE / FALSE) The probe will have the same sequence as the DNA template strand (the one you used to make the mRNA)
MB: In order to insert a human gene into a plasmid, both must ____________
be cut by the same restriction enzyme, resulting in the formation of complementary sticky ends
MB: What enzyme forms covalent bonds between restriction fragments?
MB: Using DNA technology to recombine and copy genes
1. Restriction enzyme is used to cut open a plasmid
2. Plasmid serves as a cloning vector
MB: The process by which a bacterium takes up a plasmid from the surrounding solution
MB: Occurs with the replication of a recombinant plasmid
MB: The unpaired nucleotides produced by the action of restriction enzymes
Sticky ends (they will "stick" to a complementary single-stranded sequence)
MB: Matching sticky ends
The matching of sticky ends follows the rules of specific base pairing
MB: In gene-cloning projects, plasmids function to ________
allow replication of the DNA that's being cloned
MB: To isolate a cloned gene from a DNA library, researchers ______
probe the library using a labeled single-stranded DNA complementary to the gene
MB: What is the difference between a gene (genomic) library and a gene clone?
A genomic library contains many different DNA sequences; a gene clone contains one type of DNA sequence
MB: If a scientist needs to put new DNA into a bacterium, she must _____ the bacterium
MB: In recombinant DNA methods, the term vector can refer to _____
A plasmid used to transfer DNA into a living cell
MB: What information can not be obtained from the sequence of a gene?
Whether the gene is methylated
MB: What is the polymerase chain reaction (PCR)?
A method to amplify a fragment of DNA
MB: True or false? Comparison of the sequences of the same gene across species can give some insight into the existence of a common ancestor with that gene.
MB: True or false? The Taq enzyme is a type of DNA polymerase that allows researchers to separate the DNA strands during the annealing step of the PCR cycle without destroying the polymerase.
MB: How many DNA molecules would there be after four rounds of PCR if the initial reaction mixture contained two molecules?
MB: During which step in the PCR cycle are nucleotides used?
MB: During which step in the PCR cycle do primers form bonds with a single-stranded template?
MB: Why is "chain reaction" an appropriate part of the term PCR?
Newly synthesized DNA segments serve as templates in subsequent cycles
MB: What controls which DNA is amplified in PCR?
The choice of primers
MB: Each round of PCR ______
doubles the amount of DNA
MB: What is a primary difference between polymerase chain reaction (PCR) and traditional cloning procedures such as those used to clone the human growth hormone gene?
PCR eliminates the need for restriction enzymes, vectors, and cells
MB: What information is critical to the success of polymerase chain reaction (PCR) itself?
The DNA sequence of the ends of the DNA to be amplified must be known
MB: In a single polymerase chain reaction (PCR) cycle consisting of 15 seconds at 94°C, 30 seconds at 50°C, and 1 minute at 72°C, what is happening in the step run at 50°C?
Primers are annealing to the DNA to be amplified
Removing DNA sequences from an organism, manipulate them, and insert them into different individuals
Role of enzymes in genetic engineering
- cut DNA at specific sites
- paste DNA sequences together
Recombinant DNA technology
techniques used to engineer genes
Pituitary gland produces HGH
- stimulates growth
- codes for GH1 gene
- 191 amino acids
Pituitary dwarfism (slower growth / shorter stature)
Pituitary Dwarfism (Type I)
Autosomal recessive trait (2 copies of defective gene)
Initial treatment of dwarfism
Injections of naturally produced growth hormone
Were natural injections successful?
Only successful when treated with GH from human proteins
Infectious proteins causing degenerative brain disorders in mammals
Developed in children treated with HGH therapy
Source of prion disease
Insert fully functional copies of human GH1 into E. coli to produce huge quantities of recombinant progeny
Catalyzes the synthesis of DNA from RNA template
DNA produced from RNA
Purpose of cDNA
Researchers add a chemically synthesized primer to a single-stranded cDNA and used DNA polymerase to synthesize the second strand
Isolate mRNA's from...
...pituitary gland cells and use enzymes to reverse-transcribe mRNA→cDNA
Product of reverse transcription
Double-stranded cDNA corresponding to each gene actively expressed
Producing many copies of a gene
How to clone?
Insert a gene into a small, circular DNA molecule (plasmid)
Replication (passing on plasmids)
Splice loose DNA into a plasmid and insert it into a bacterial cell hoping the bacteria grow/divide
Using a plasmid to make copies of a foreign DNA sequence
Bacterial enzyme to cut DNA molecules at specific base sequences for later insertion into a cloning vector
Word/Sentence that reads the same way backward as it does forward
5'→3' is identical to the 5'→3' sequence on the antiparallel, complementary strand
Inserting Genes into Plasmids
1. Identify a palindromic recognition site
2. Add restriction endonuclease
3. Sticky ends result
4. Insert gene into plasmid
1. Identify a palindromic recognition site
- plasmid contains a recognition site for a restriction endonuclease
- attach same recognition site to cDNA corresponding to a gene that will be inserted into the plasmid
2. Add restriction enzyme
Makes staggered cuts at each of the recognition sites
3. Sticky ends result
Recognition sites now have "sticky ends" capable of H-bonding with a complimentary sequence
4. Insert Gene into Plasmid
Sticky ends on plasmid and on gene to be inserted bind by complimentary base pairing. DNA ligase catalyzes formation of a phosphodiester bond at points, "sealing" the inserted gene
Recombinant DNA technology
The ability to create novel combinations of DNA sequences by cutting specific sequences and pasting them to new locations
If a recombinant can be inserted into a bacterial/yeast cell, foreign DNA will be copied and transmitted to new cells as the host grows/divides
Cells that take up DNA from the environment and incorporate it into their genomes
How does one increase plasma membrane permeability?
Chemical treatment/electrical shock
- Collection of DNA sequences, each of which is inserted into a vector
- provide a way to store DNA fragments from a particular cell type or genome in a form that is accessible for gene cloning
The sequences are cDNAs made from a particular cell type or tissue
The sequences are fragments of DNA from an individual's genome
single-stranded fragment that will bind to a particular single-stranded complimentary sequence in a mixture of DNA
- by binding to the target sequence, the probe marks/distinguishes the fragment containing that sequence
Create a cDNA library
1. Isolate mRNAs (from cells in pituitary gland)
2. Synthesize cDNA from each mRNA using reverse transcriptase
3. Make cDNA double-stranded using reverse transcriptase (or DNA polymerase)
4. Make recombinant plasmid: insert each cDNA in a different plasmid
- introduce recombinant plasmids into E. coli cells by making cells permeable to DNA
- Each cell contains one type of recombinant plasmid and thus one type of cDNA... collection of cells = cell library
Using a DNA Probe
1. Make probe (single-stranded DNA probe has a label that can be visualized)
2. Expose probe to a collection of single-stranded DNA sequences
3. Find probe... it binds to complementary sequences in target DNA (now labeled and ready to be isolated)
Screening a cDNA library
1. Grow transformed E. coli cells containing plasmids on many plates (each colony = different cDNA)
2. Lay a filter on each plate, then remove... some cells from each colony stick to the filter
3. Treat bacteria with chemicals to break open cells and make DNAs single stranded
4. Probe filters with labeled DNA
5. Find probe binding to the complementary sequence in the cDNA library
6. Identify colony (on original plates, find colony of E. coli cells containing growth hormone gene)
How to use the genetic code to predict possible DNA sequences of GH1?
- deduce a set of possible sequences that could encode the GH1 gene
- chemically synthesize set of short, single-stranded DNAs that were complementary to the possible GH1 sequences
Curing dwarfism with recombinant DNA
The promoter sequence is recognized by...
...RNA polymerase holoenzyme
Polymerase Chain Reaction... an in vitro DNA synthesis reaction that uses DNA polymerase to replicate a specific section of DNA over and over
Since sequence information is required for PCR...
...start by synthesizing short lengths of single-stranded DNA, matching sequences on either side of a gene
Once primers are bound...
...DNA polymerase extends each new DNA strand 5'→3'
1. Start with a soln containing template DNA, primers, Taq polymerase, and an abundant supply of the 4 dNTPS
3. Primer annealing
5. Repeat 2-4
6. Repeat the cycle 20-30 times
Heating separates strands in double helix
At lower temps, primers bind to template DNA by complementary base pairing
Taq polymerase uses dNTPs to synthesize complimentary DNA strand, starting at primer
When steps 2-4 are repeated, what happens?
The copies of DNA double
True or False? Taq polymerase is heat stable
Which steps constitute a single PCR cycle?
Denaturation, primer annealing, and extension
A total number of "n" cycles can generate __________ copies
So old... most DNA had degraded to tiny fragments
Determining if modern humans shared sequences with Neanderthals
- design primers to bracket a region
- produce millions of copies of Neanderthal DNA fragments (sequence entire genome of 3 Neanderthals)
Which if he following lists the steps of creating s human brain cDNA library on the proper order
Isolate mRNAs, synthesize cDNA, Make recombinant plasmid, transform into cells
If mRNAs could be lighted and replicated within plasmids, what enzymes commonly used in recombinant DNA technology would no longer be needed
Which of the following would NOT be true of cDNA produced using human brain tissue as the starting material
It could be modified for use as a probe to detect genes expressed in the brain
Many identical copies of genes clones in bacteria are produced as a result of
Plasmid and bacterial cell replication
In recombinant DNA methods, the term vector can refer to
A plasmid used to transfer DNA into E. Coli
The difference between a gene library and a gene clone is that a gene library...
...contains many different DNA sequences while a gene clone contains one
A gene library from a particular humans retinal cell would be a collection of
Genes that have been sequences from a particular organism
How do we describe transformation in bacteria
Uptake of external DNA into a cell
Which of the following would be most useful for increasing he amount of DNA available for testing
What is a primary difference between PCR and tradition cloning procedures such as those used to clone the HGH gene
PCR eliminates the need for restriction enzyme, vectors and cells
What information is critical to he success of polymerase chain rxn itself
DNA sequence of the ends of DNA to be amplified must be known
In a single PCR cycle what is happening in the step run at 50•C
Primers are annealing to the DNA
Which of the following is in the correct order for one cycle of PCR
Denature DNA, anneal primers, extend primers
Which of the following dsDNA sequences in is most likely to be recognized as restriction enzyme cutting site
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