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33 Cards in this Set

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What are restriction endonucleases and what do they do?
-bacterial restriction enzymes
-cleave DNA at specific sequences
> Recognize UNIQUE DNA sequences 4-8 base pairs long
>Cleave phosphodiester bonds within or adjacent to the recognition sequence
>Restriction enzyme sites are usually PALINDROMES
(same way forward as it is backwards)
> frequency of sequence=1/ (4^n) where n=number of base pairs in the recognition sequence
> creates ends that can easily come together and are sealed by DNA ligase
What do you use Restriction Endonuclease for?
to make specific proteins (ex. insulin)
What are the steps in making more DNA and making proteins.
-DNA that was cut by restriction enzyme is inserted into bacterial plasma.

- The plasmid contains a bacterial resistance gene to distinguish cells with the plasmid from those without. (Cells treated with a chemical, those that survive have the plasmid)

-Plasmids can be constructed to make useful proteins like insulin. (Must use reverse transcriptase on mRNA to make sure introns are removed in order to accurately reproduced in a bacterial cell)
What must be used to make sure introns are removed in order to accurately reproduced in a bacterial cell.
reverse transcriptase
What is the main function of Agarose Gel Electrophoresis?
separate nucleic acids by size.

[helps us to know that the DNA is cut at the right place by the restriction enzymes]
What are the steps of Agarose Gel Electrophoresis?
1. DNA is negatively charged, the molecules move in the field toward the positive end of the circuit.
2. SMALLER DNA fragments move through the gel QUICKER.
3. Ethidium Bromide slips in between the bases of DNA and fluoresces under UV light.
What is the main function of Southern Blotting?
detects large delections, insertions, and sequence rearrangements in DNA
What are the steps of Southern Blotting?
1. Cleave (cut) DNA with restriction enzyme.
2. (Gel treated with alkalai solution to separate strands of DNA (denature DNA)
3. Separate fragments by size in agarose gel.
4. Soak in NaOH to neutralize pH to 7.
5. Apply nitrocellulose membrane and paper towels on top (for capillary action)
6. Mix membrane and radioactive probe to check for targeted DNA sequence.
(Have to know what y
you’re looking for to make the probe)
What are some facts about the probe used in southern Blotting
-Radioactive piece of DNA
(oligonucleotide)
-Matches a section of one (or more) of the bands on the gel
What can southern blot be used for?
-Semi-quantitative since amount of probe hybridized is proportional to the amount of target DNA sequence on the membrane.
-Southern Blot can be used to develop information on size and organization (rearrangements) of a gene
-DOES NOT detect small changes
-Sensitivity
PCR > Southern Blot > Gel electrophoresis
What is the Northern Blot used for?
-Used to determine size of mRNA
-Used to determine amount of mRNA
-Used to determine splice variants (alternate splice forms) of mRNA
-Used to compare expression levels (expressed vs. not expressed)
-Used to detect altered gene transcripts caused by insertions or deletions.
What is Karyotype Analysis used for?
-for DNA
-Direct visualization of whole chromosomes
-uses DNA probes
-Detect large changes in chromosome structure (deletion, rearrangements, amplification) and changes in chromosome number
What is used for detection in Karyotype Analysis
fluorescent in situ hybridization (FISH) uses a fluorescent dye

used to use Giemsa dye but it binds more to A-T content then G-C
When would karyotype analysis be most useful?
Pre/post natal. Ex. Down syndrome
What is DNA Sequence Analysis and what does it involve?
Determining the sequence involves
>random termination of growing complementary strands
>terminates at every possible position,
>but terminates on different individual molecules in the population of newly-synthesized strands.
-Di-deoxy bases are used to terminate the growing complementary strand.
-Sanger is the older method (first generation sequencing)
-Second generation sequencing technology is faster and cheaper
-determine the sequence of the complementary strand
What is the procedure for PCR Cycle?
1. Heat to denature
2. Cool to hybridize
3. 72 degrees C to allow DNA polymerase to work
4. Repeat
What is the importance of the PCR Cycle
-Highly specific
-Highly sensitive
-Widely applicable (know what it’s used for)

Allows for a million fold amplification of the tiniest amount of DNA.
What is the purpose of Microarray analysis?
-look at all of mRNA not just one as in N. blotting
-To analyze the level of expression of all the expressed genes (as mRNA) in a cell
-Used to classify cancers
-A large collection of individual probes corresponding to the set of known genes is spotted on a microscope slide


overexpressed in tumor = red
repressed in tumor = green
No change in expression = yellow
What type of gel is used for electrophoresis of proteins?
-Sodium dodecyl sulfate denatures proteins into rod-like shapes and it simultaneously coats them with a uniform, negative charge.
-Mercaptoethanol breaks disulfide bonds in proteins.
What is Western blotting?
-Similar to Northern/Southern
-An antibody binds a particular protein
-Then another antibody binds the original antibody. The second antibody is tagged with some marker.
-Used to detect protein expression
-Can also detect covalent modifications such as phosphorlyation.
What is the frequency of cleavage sites in a DNA molecule inversely proportional to?
the length of recognition seqence

frequency=1/4^n
where n= number of bases in recognition sequence
What type of membrane is used in Southern Blotting?
nitrocellulose membrane
What are Northern blots used to detect?
Detects alternate spliced forms of mRNA (different sizes)

asses gene regulation (expressed/not expressed)

complare expression levels
At what concentration of chain-terminating (ddNTP) nucleotides is synthesis carried out?

[Sanger's chain termination]
in the presence of low concentration of chain-terminating (ddNTP) nucleotides
What is the purpose of Sanger's chain termination sequencing method?
To determine a DNA sequence

The automated Sanger method helped to determine the complete sequence of the human genome
What is the purpose of (polymerase chain reaction) PCR?
Powerful method for detecting and quantitating DNA or RNA.
What is the purpose of gene microarrays?
genome wide expresson analysis.
Many genes (as mRNAs) quantitated simultaneously


Emphasis on global changes in expression instead of changes in single genes.
What is the importance of microarrays?
Understanding disease
Distinguishing disease subtypes
Identifying new diagnostic markers
Revealing new therapeutic targets
How does the SDS Gel Electrophoresis separate proteins?
shape and charge
How do you separate proteins based on size alone?

[SDS Gel Electrophoresis]
Sodium Dodecyl Sulfate (SDS) detergent is used
What is used in Sodium Dodecyl Sulfate (SDS) Gel Electrophoresis to break disulfide bond crosslinks?
mercapto-ethanol
What is the benefit of using bacteria for replication of desired gene?
Avoids expensive and sometimes dangerous purification
Why would mammalian cells be used?
When proteins acquire normal post-translational modifications, e.g., phosphoylation