Term
| What are the basics of restriction endonucleases? |
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Definition
Recognize groups of bases (motifs), from 4-8, in a specific manner.
Sequences arranged in palindromes.
Make double stranded breaks.
Many of these enzymes generate DNA fragments with “sticky ends”.
Also allow highly specific maps of the organismal genome to be made.
“Sticky ends” allows unrelated pieces of DNA to be put together. This is relevant to cloning foreign DNA. |
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Term
| What are the different type of vectors used in cloning? |
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Definition
Plasmid – circular DNA molecule that can replicate in bacteria
Cosmid – vector based on bacteriophage gamma
BAC – bacterial artificial chromosome (F-factor plasmid)
PAC – P1 artificial chromosome (P1 bacteriophage)
YAC – Yeast artificial chromosome (Yeast chromosome
replication start)
The different vectors are used for different DNA sizes. For example one cannot fit the dystrophy gene in a plasmid |
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Term
| What are the basics of a plasmid? |
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Definition
It is an autonomously replicating bacterial minichromosome.
Carry antibiotic resistance genes: which can be used to select for those bacteria carrying that plasmid [Marker]. |
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Term
| What is insertional inactivation? |
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Definition
This is when one uses a restriction endonuclease to create a recombinant plasmid. The gene to be cloned is inserted into a region on the plasmid that confers antibody resistance and this resistance is now lost.
This allows for separation of bacteria with the desired plasmid |
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Term
| What are the primary components of the cosmid vector? |
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Definition
The MCS or multi cloning site with a different number of unique restriction endonuclease sites
the bacteriophage piece
the eukaryotic virus vector and the vector for the drug that kills the eukaryotic virus (used in selection of desired cosmid) |
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Term
| What is an expression plasmid? |
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Definition
This is a plasmid where the cDNA coding region has been inserted
Translation creates a fusion protein that includes the normal bacterial amino acids plus the foreign gene product from the inserted cDNA
Antibodies are used to select for the fusion proteins |
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Term
| How does antibiotic screening of plasmids work? |
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Definition
After the recombinant plasmids are created and inserted into E coli cells, the cells are grown on a plate with ampicillin.
Only the E coli with the recombinant plasmid (which contains the ampicillin resistant gene) will survive and thrive |
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Term
| What is the RT-PCR method of gene cloning? |
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Definition
1) isolation of mRNA
2) reverse transcription of mRNA to produce the coding DNA
3) PCR of the coding DNA used for amplification
This eliminates all unexpressed genes, focusing only on those genes that are expressed in that cell line
This method can be used to create a cDNA library of expressed genes |
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Term
| What is an expression library? |
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Definition
This is where the cDNA's produced from RT-PCR are cloned into a bacterial expression vector.
The probe used is an antibody probe |
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Term
| What is a genomic library? |
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Definition
This is where the full complement of the genome is cloned.
A nucleic acid probe is used for the genomic library as well as the cDNA library |
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Term
| What are the different types of hybridization? |
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Definition
Hybridization is used to identify the cloned fragment of interest
- stringent hybridization uses a very specific synthetic DNA probe (complementary to the fragment of interest) and only double stranded helices will form
- reduced stringent hybridization uses a less specific DNA probe (unsure about the specificity) and several different complexes can form that aren't fully complementary |
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Term
| What is the purpose of Southern Blotting? |
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Definition
Cleavage of a specific DNA with a RE yields a very
distinct pattern of RE fragments, that can be separated
by gel electrophoresis
Southern blotting is used to separate different sized restriction endonuclease fragments of DNA.
It is used clinically in mutations that affect RE sites |
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Term
| How are Northern and Western blotting related to Southern blotting? |
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Definition
Northern blotting is very similar to Southern blotting except that it uses mRNA as opposed to DNA
Western blotting uses the same principles as well but it uses proteins and uses antibodies as probes as opposed to specific DNA probes |
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Term
| Explain how Southern blotting could be useful in the diagnosis of mutations |
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Definition
If there are mutations that affect the restriction endonuclease sites then the RE is no longer going to be able to cut at that site.
This will alter the number and/or the size of the fragments
Southern blotting allows one to separate the different fragments based on length. The results will tell you the size of the fragments and whether you have different sized fragments.
This information can be used to determine the genotype - homozygous normal, heterozygous, or homozygous recessive |
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Term
| How can allele-specific probes come into play during Southern blotting? |
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Definition
One can do Southern blotting with two separate lanes each with its own DNA specific probe. One lane will contain a specific probe for the normal DNA sequence and the other the probe for the mutated DNA sequence.
Fragments only in Lane 1 indicate homozygote normal
Fragments only in Lane 2 indicate homozygote recessive
Fragments in both lanes indicate heterozygote
The probes are allele-specific and the mutation must be known for this to work |
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Term
| What are restriction length fragment polymorphisms? |
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Definition
These are polymorphisms caused by a base substitution in the recognition site of a restriction endonuclease
The mutations cause a change in the length of the fragments created by restriction endonucleases
The changes can be detected by Southern blotting |
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Term
| What is variable number of tandem repeats? |
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Definition
There are segments of DNA that have repeat sequences called tandem repeats
The tandem repeats can occur in fragments that are detected by probes. The mutant fragment will have a different number of tandem repeats from the normal fragment and this causes the fragments to be different lengths.
Both PCR and Southern blotting can be used to detect changes in the # of repeats.
used in DNA fingerprinting |
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Term
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Definition
A simple technique used to diagnose mutations.
It uses separate fluorescent-labeled probes for the normal sequence and the mutated sequence
Denatured DNA is exposed to the normal probe in one dot and the mutant probe in the other dot
Positive for:
only normal probe = homozygous normal
both probes = heterozygous
only mutant probe = homozygous mutant |
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Term
| How is DNA sequencing controlled? |
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Definition
The controlled interruption of DNA synthesis using dideoxynucleotides.
These nucleotides bind to their complementary nucleotide and terminate the chain (b/c of the dideoxy)
The four different dideoxynucleotides will create different fragments based on where they terminate
The fragments will run on a gel (separate lane for each dideoxynucleotide used) and the info is then used to determine the sequence of the original DNA |
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Term
| What are the two different methods of creating transgenic organisms? |
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Definition
The older method is to inject DNA into fertilized oocytes in vitro and then re-implant several oocytes back into the mother
The newer method is to culture embryonic stem cells and inject them into blastocysts and re-implant the blastocysts. |
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Term
| What are the basics behind pharming? |
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Definition
1) insertion of desired gene into an expression vector
2) growing colonies containing desired gene
3) removing nucleus from oocyte
4) taking nucleus with desired gene and inserting into the enucleated oocyte
5) stimulation of cleavage and implantation of blastocyst creating a transgenic organism which will produce the desired protein |
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Term
| What is the role of antisense RNA in gene technology? |
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Definition
1) production of functional mRNA from DNA sequence that lies between two restriction sites
2) the mRNA is used to produce cDNA which is inserted backwards into a recombinant expression vector
3) transcription of this vector creates antisense RNA
4) the antisense RNA can hybridize with normal (sense) RNA and block its translation |
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Term
| What are the basics of PCR? |
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Definition
PCR is polymerase chain reaction
In this reaction there is a forward primer and a reverse primer specific for the sequence that needs to be amplified
Many cycles of denaturation, hybridization, and ligation cause many identical sequences to be produced from the original ds DNA |
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Term
| How is PCR used clinically? |
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Definition
PCR can be used to scan for exon deletion
If an exon is deleted then PCR for that exon will produce no product. with the use of a control, this result indicates a deleted exon on both alleles. |
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