Tools of Molecular Genetics Flashcards
Cut DNA molecules at specific sites
-Reckognizes short sequences of double stranded DNA up to 4-8 bps
Restriction Nucleases
Recognize palindromic (read the same 5’ to 3’) sequences
Restriction Nucleases
Once restriction enzymes recognize the pallindromic sequences, they cut in which two ways?
- ) Blunt ends
2. ) Sticky ends
A cut at the center of the recognition sequence results in
Blunt ends
The sticky end cuts are very useful when we are trying to
Clone DNA
RNA gel electrophoresis is similar to DNA gel electrophoresis with what exception?
RNA forms extensive secondary structure
The extensive secondary structure that RNA forms prevents it from migrating strictly according to its size. How do we get around this?
Add denaturants such as formamide
What do we use to visualize the electrophoresis gels of DNA and RNA?
Ethidium bromide staining or radioisotope labeling
To visualize the DNA bands, the gel is soaked in a dye (such as ethidium bromide) that binds to DNA and fluoresces brightly under
UV light
An alternative method for visualizing DNA is
-The exposure of x-ray film to a radioactive sample
Autoradiography
DNA can be labeled with radioactive isotope
-Will expose autoradiographic film
P32
Allows us to compare and analyze DNA and RNA molecules of identical or related sequences
Hybridization
Advantageous for detecting specific species of DNA or RNA in a mixture and estimating the quantities of each
Hybridization
The single stranded DNA or RNA used to detect the unknown DNA is called a
Probe
How can we denature the double stranded DNA or folded RNA so that we can hybridize with our probe?
Denature with either High temp or High pH
Once we add the probe, we can allow the DNA to reannel by
Lowering the temp or pH
We pick conditions to ensure that DNA or RNA anneals with our probe. How well these conditions allow our DNA or RNA to anneal together is called
Stringency of conditions
The higher the stringency, the
Lower the probability of hybridization
Increasing the temperature or amount of denaturing agent such as formamide raises the
-Lowers probability of hybridization
Stringency
If we want only a very specific fragment to anneal with a probe, we can adjust conditions accordingly. For example, we could
Raise temp or concentration of formamide
If we want to anneal heteroduplexes with mismatches, we can use
Lower temp or lower conc. of formaldehyde
Another way to manipulate stringency is through the length of the
Probe
Form stable heteroduplexes with target sequences that are similar but not identical to the probe
Longer nucleic acid sequences (more than 100 nucleotides long)
Allows cross-species analyses and identification of distantly related members of a gene family
Reduced stringency
Less tolerant of sequence mismatches than long probes
Short probes
Makes it so that it is possible to select for perfectly matched duplexes only
Short probes (High stringency)
Long probes can form stable hybrids even in the presence of
Mismatches
Allow you to distinguish between allelic sequences that differ by just a single nucleotide
Short probes (High stringency)
An inherited difference in the pattern of restriction enzyme digetion
Restriction Fragment Length Polymorphism (RFLP)
In order to detect RFLP we need to use
Restriction fragments
The target molecule of interest in southern blotting is
DNA
Used to detect specific DNA fragments
Southern Blotting
Useful in investigating the number of copies of a gene or whether there are large deletions, insertions, or rearrangments
-Can also be used to detect a point mutation
Southern Blotting
Describe a southern Blot
- ) Unlabeled DNA cut w/ restriction enzyme
- ) DNA fragments separated by agarose gel electrophoresis
- ) Fragments blotted onto nitrocellulose paper
- ) Labeled DNA probe hybridized to separated DNA
- ) Sheet is washed so that only hybridized DNA fragments remain
- ) Labeled hybridized fragments visualized by autordiography
An adaptation of southern blotting to detect specific sequences in RNA
Northern Blotting
The radioactive probe for Northern Blotting is usually a
Single stranded cDNA molecule
Allow us to monitor gene expression levels
Northern Blots
More efficient way than a Northern Blot to monitor gene expression levels
DNA microarrays
Used to monitor the expression of many thousands of genes simultaneously
DNA microarrays
How do DNA microarrays work?
Reverse transcribed mRNA is turned into cDNA labeled with fluorochrome and hybridized to a microarray. The colored spots on the microarray will show which gene is expressed at a higher level
Designed to match the nucleotide sequence flanking the substitution
Allele-specific Oligonucleotides (ASOs)
Patient DNA is hybridized to a panel of mutation specific probes
Allele specific Blotting
Allele specific blotting is used to detect things like
Sickle cell mutations and as a strategy for screening for thalassemias
Simple and rapid technique to amplify the sequence of a target DNA up to 10^9 fold in just a few hours
Polymerase Chain Reaction (PCR)
A valuable tool in medicine because it allows for the ampliphication of even trace amounts of DNA
PCR
PCR is a repetition of many cycles consisting of three steps. What are the three steps?
Step 1.) Heat to separate DNA strands
Step 2.) Cool and add primers (annealing)
Step 3.) Primers are incubated w/ DNA polymerase and the four deoxynucleotides, and complementary strands are synthesized
In order to perform PCR we need to design two
Primers (one for each strand)
Direct the amplification of the desired piece of DNA
PCR Primers
PCR depends on usage of a heat stable
Polymerase (taq polymerase)
What amplification does each cycle of the PCR have?
Each cycle doubles the number of strands from the previous cycle
Uses repeat rounds of strand separation, hybridization, and synthesis to amplify DNA
PCR
Dependent on perfect base-pairing of the 3’ end of nucleotide primers
Allele specific PCR
Allows us to selectively amplify a mutant allele cause by a mismatch
-because the designed primer will bind to the mismatch much better than taq polymerase
Allele specific PCR
mRNA is isolated from a tissue, and then a cDNA is synthesized using a reverse transcriptase. The original RNA template is removed by RNAse H and cDNA is amplified. PCR then occurs
Reverse Transcriptase PCR (RT-PCR)
Removes the RNA tamplate in RT-PCR
RNAse H
In this technique, a target DNA strand is replicated in vitro using a primer, DNA polymerase, and a mixture of dNTPs for adenine, guanine, thymine, and cytosine in four different reaction tubes. Each reaction tube also contains a small amount of one of the four radiolabeled nucleotide analogs
DNA Sanger sequencing
How does Sanger sequencing work?
Randomly, the radioactive nucleotide analogs will be inserted into the newly synthesized strand and synthesis will stop. This allows us to see which nucleotide occupies which position in the target DNA
The nucleotide analogues lack the
3’ OH needed for synthesis to continue
When the Sanger sequencing reaction is complete, each tube contains a mixture of radioactively labeled
DNA fragments of different length
The bottom of the gel for Sanger sequencing represents the
5’ End of the newly synthesized DNA
The top of the gel for Sanger sequencing represents the
3’ end of the newly synthesized strand
The strand synthesized by Sanger sequencing will be complementary to our
Target strand
A single reaction tube will contain the target DNA, a primer, all four dNTPs and all four analogues
Automated DNA sequencing
In automated DNA sequencing, the PCR reaction will generate the DNA fragments that are separated by capillary electrophoresis. The fluoresence emission of each peak is monitored by a laser detector and recorded on a chromatogram where the 5’ end of the sequence is on the
Left
Recombinant DNA can be copied inside of
Bacterial Cells
Some bacteria can efficiently take up foreign DNA from their surroundings, a phenomenon called
Transformation
Plasmids that are taken up by the host bacteria are maintained as a piece of DNA independent of the bacterial chromosomes. Therefore, they are useful tools because their replication is independent of the
Host Cell
Specialized plasmid vectors are used to
Clone DNA
How do we clone DNA using plasmids?
Cut open circular plasmid and insert the DNA fragment to be cloned. When the resulting recombinant DNA replicates, our fragment will be cloned
Cleaves the circular double-stranded plasmid (vector) DNA for insertion of the DNA fragment we want to clone
Restriction Nuclease
The DNA fragment we want to clone is covalently linked to the vector by
DNA ligase
The DNA fragment we wish to clone must be cut with the same
Restriction enzyme
The recombinant plasmid is made inside of a test tube. When we introduce the recombinant plasmid DNA into a bacterial cell, what happens?
The plasmid will be replicated millions of times
We can use DNA cloning by plasmid vectors to compile a
DNA library
A collection of cloned fragments of an organism
- Two types
1. ) Genomic
2. ) cDNA
DNA libraries
Libraries of human genomic DNA fragments can be constructed using
Restriction nuclease and ligase
A bacterial colony carrying a particular DNA clone can be identified by
Hybridization
When compiling a genomic library, once we have transferred our bacterial colonies to a sheet, we can lyse the bacteria and denature the DNA with
Alkali
After we have denatured the DNA, we then add radioactively labeled DNA probes specific for the plasmid of interest. Then we expose the paper to
Photographic film
Living cells containing the plasmid of interest can then be isolated from the colony disk and
Grown in large quantities
Represent the mRNA produced by a particular tissue
cDNA libraries
c = complimentary
cDNA is double stranded DNA synthesized from RNA by
Reverse transcriptase and DNA polymerase
Genomic DNA clones and cDNA clones derived from the same region of DNA are
Different
Exons, introns, and non-transcribed DNA are included in the DNA clones of the
Genomic Library
The intron sequences are removed and a continuous coding region is present in each clone of the
cDNA library
Allows you to see which genes are expressed more frequently
cDNA Library
Genes will be represented equally regardless of their levels of expression in the
Genomic Library
Can be used to splice together a set of DNA fragments derived from different sources (making Chimeras)
Serial DNA cloning
Why do we need to make chimeric proteins?
Adding a fluorescent protein to a protein of interest allows us to visualize where the protein is located in the cell
In serial DNA cloning, after each DNA insertion step, the recombinant DNA is
Cloned (purifies sample)
The purified cloned DNA is then cut with a restriction nuclease, and
Another DNA fragment is added
Can be produced from a protein-coding sequence cloned into and expression vector and introduced into cells
Large amounts of protein
To use DNA cloning to make a protein, we first must make a vector plasmid containing a
Highly active promoter for the protein of interest
Make it possible to move experimentally from gene to protein and from protein to gene
Recombinant DNA techniques
If we want to make DNA from a protein, we must determine the amino acid sequence of a purified peptide fragment, then we want to
Search the DNA database for the gene sequence
Used to determine the pattern of a gene’s expression
Reporter Genes
Let’s say we want to find out which cell types express protein “X” but it is difficult to detect the protein “X” directly, what can we do?
Replace the coding sequence for protein “X” with a reporter gene that expresses a protein like GFP which can be easily monitored
The reporter protein will only be expressed in places where
The target protein is expressed
In order to tell which regulatory regions control expression in particular cell types, we
Turn off all but one sequence at a time and see where the protein is expressed
We can add epitopes as
Reporters
In order to create an epitope tagged protein, we fuse the gene for the epitope tag to the gene for the
Target Protein
We can use DNA cloning and recombinant DNA technologies to genetically modify animals by
Gene replacement, gene knockout, and gene addition
When the normal gene is entirely replaced by a mutant copy of the gene
-provides information on the activity of the mutant gene without interference from the normal gene
Gene replacement
The normal gene can be completely inactivated by making large deletions in it. This process is called
-Widely used to obtain information on the function of the normal gene
Gene knockout
A mutant gene can be added to the normal genome. This provides information when the introduced mutant overrides the function of the normal gene
Gene addition
