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Three major technological breakthroughs have revolutionize biology
Restriction endonulceases – allows us to cut DNA into manageable fragments.
The technology which allowed us to make huge amounts of the DNA fragments (PCR)
Technologies that allowed us to know when we had gotten the DNA fragment for which we were looking.
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Restriction endonucleases are
Is an enzyme that cuts DNA at specific site
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A Palindrome is
Sequence of units that can be read the same way in either direction
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The type II restriction endonuclease
Cuts at a precise sequence of DNA that is a palindrome
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A methylase does what to the nucleotides in a sequence
Methylates one or more of the nucleotides in the sequence and protects the bacterial DNA from being cut by a virus
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What is the specificity of TaqI and HaeIII restriction endonucleases
- HaeIII- cleaves the DNA at the positions where the GGCC sequence is found.
- TaqI cuts the T off 5'TCGA and 3'AGCT
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Recombinant DNA
Is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together through the process of gene splicing
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Two pieces of DNA with the same “sticky” ends could be ligated together using
DNA ligase
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Cloning vectors are
A small piece of DNA into which a foreign DNA fragment can be inserted
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Cloning vectors allow us to
Cut up DNA from an organism, ligate it to a restriction site that has been generated by cutting the vector with the same restriction enzyme.
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In addition to the cloning site, the cloning vector has
An origin of replication, and a selectable marker that permits one to know when the vector contained an insert
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A cloning vectors can be “transfected” into a bacteria allowing the bacteria to
Make millions of copies of the cloned DNA for you.
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A DNA molecule into which foreign DNA may be inserted and which can be returned to and replicated within a living cell is called a
Vector
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To get around the intron problem and get only the coding region of a gene, you have to start with material that does not have introns such as
mRNA
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What is the sequence of events for using RNA for cDNA (Complimentary DNA)
First purify the RNA from a cell that expresses the gene of interest.
Next reverse transcribe the mRNA into single stranded DNA.
Then make it double stranded.
Finally ligate the dsDNA into a vector and search for the gene of interest in the clones you have produced.
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Dideoxynucleotides, or ddNTPs are what, and useful for
Nucleotides lacking a 3'-hydroxyl (-OH) group on their deoxyribose sugar. Making it useful for sequencing DNA because after being added, it can't form any more nucleotides
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Complementary DNA or cDNA is made in vitro using the enzyme reverse transcriptase and ____ as the usual template
mRNA
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Prokaryote Expression vector is
A plasmid that is used to introduce a specific gene into a target cell
Used to produce a lot of a protein that is encoded by the target gene
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Didanosine (dideoxyinosine, ddI) is a nucleoside analog sometimes used to treat HIV infections. This drug is converted metabolically to 2’,3’-dideoxyATP (ddATP), which blocks DNA chain elongation when it is incorporated into viral DNA synthesized by reverse transcriptase. Why does DNA synthesis stop?
There is no 3’-hydroxyl group to form the next phosphodiester bond.
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What must you include in a expression vector
The necessary elements that a bacteria needs for gene expression
- a bacterial promoter placed 5’ of the gene.
- a Shine-Dalgarno sequence place at the right place in the 5’ UTR so that the start codon will be placed in the P site of the ribosome.
- a transcription termination sequence placed after the gene.
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The only way to prevent DNA from being cut is by
Methylating it
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Which is more efficient HaeIII or TaqI
TaqI because the ends that were cut are complimentary making putting them back together again easier
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In the following figure, the first 10 bases of the DNA template is being used in this sequencing reaction are:
5'-ACGCATTCAC-3'
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A researcher has cloned and isolated a small fragment of single-stranded DNA. She sequences this fragment using the Sanger dideoxy method. The results from her sequencing gel are shown below. What is the sequence of her original single-stranded fragment?
CTGGTA
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A probe is a nucleotide sequence that is
Complementary to a specific DNA sequence
- One base difference will affect the ability of the probe to bind to the DNA.
- This allows you to discriminate between normal and mutated DNA.
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What oligonucleotide probe would best hybridize with the DNA sequence 5'-AAAAGGTTCC-3'?
-5'-GGAACCTTTT-3‘
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An allele is
One of two or more forms of the DNA sequence of a particular gene
A sequence of DNA that is almost the same as the corresponding DNA region on homologous chromosome
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Allele specific oligo-nucleotides (ASO) probes are used to
- Detect different alleles
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SNPs are
(single nucleotide polymorphisms) is a DNA sequence variation occurring when a single nucleotide doesn't match its nucleotide pair
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VNTRs
(variable numbers of tandem repeats) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat
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SNPs are one of the
primary causes of diseases
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VNTRs are being exploited to
identify individuals by forensic scientists
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A common way to detect the VNTRs is to
PCR the region and then run the amplified produces on a gel.
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Chorionic villi sampling can be done when in the pregnancy compared to an amniocentesis.
Earlier
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Sometimes a mutation that is causing the disease changes a restriction endonuclease site. When this occurs, it provides
An easy way to detect the mutation because the spliced site will differ in length from the original
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Polymerase Chain Reaction (PCR)
is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude
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Polymerase Chain Reaction (PCR) requires what to work
- DNA (very little is needed)
- Deoxynucleotides (large amounts are needed)
- DNA primers that flank the region of interest
- A heat stable DNA polymerase
- The ability to quickly increase and decrease the temperature of the reaction (a PCR machine)
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Polymerase Chain Reaction (PCR) steps are
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Genetic testing for cystic fibrosis can be done by
using PCR
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Microarray analysis of gene expression allows one to investigate
- Nearly all the genes that are expressed in a cell at one time.
- Useful for making better treatments for cancer
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ELISA stands for enzyme linked immuno-sorbant assay and does what
The ELISA uses antibodies to detect proteins (including antibodies).
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The ELISA can give false-positive results because
of “cross-reactive” antibodies.
A Western-blot is usually performed to find false positives.
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A knockout mouse is
A genetically engineered mouse in which one or more genes have been turned off through a targeted mutation.
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Neomycin is a marker gene which means after you integrate into a cell
You can tell if the gene has integrated into the host DNA if the cells become resistance to neomycin.
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Gene Therapy is
- the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease
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DNA sequencing is run on what type of gel
Polyacrylamide
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Southern blots are run on what type of gel
Agarose
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DNA has what charge in it
Negative
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Southern blotting is used solely for
DNA, and detects changes in it
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DNA fingerprinting is done by what method
Southern Blot
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DNA fingerprinting refers to looking for matches in
Repeat sequences
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VNTR's and SNP's are used in what method
Southern Blotting
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PCR and DNA sequencing differ in what ways
DNA sequencing often uses the sanger method utilizing the Dideoxy-nucleoside triphosphates like ddATP, ddGTP, ddTTP, and ddCTP
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A northern blot sample analyzes what
RNA, to measure RNA amounts
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The Western blot analize what
Protein, measuring protein amounts
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ASO analyzes
DNA mutations without a gel
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Microarray analyzes
RNA and cDNA without a gel, it measures many mRNA at once
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