Chapter 8 Flashcards
How can one identify a specific gene sequence when the entire genomic sequence is present?
In 1975, E. M. Southern reasoned and showed that one could identify DNA with like (similar) DNA
The reason for this is that single-stranded DNA can base pair with its complementary strand of DNA.
This technique is termed hybridization, and the process of identifying DNA after separation by gel electrophoresis is called Southern hybridization
Summary of Southern hybridization (8 steps)
- Fractionate and separate DNA by gel electrophoresis
- Denature DNA by soaking gel in alkaline solution followed by neutralization solution.
- Transfer the DNA from the gel to a membrane
- Crosslink the DNA to the membrane
- Prehybridize the membrane with a solution that does not contain probe DNA.
- Hybridize membrane with a solution containing denatured labeled DNA probe
- Wash the membrane
- Develop the membrane
Southern Step #1
DNA fractionation/separation - In order to identify DNA from a population of DNA, the DNA can be digested by a restriction enzyme and separated by gel electrophoresis. The result of this is typically a ‘smear’ of DNA down the lane of the gel, because there are millions of different sized fragments that are produced
Southern Step #2
DNA denaturation - Before DNA can be transferred to a membrane, it is typically denatured in the gel.
This is done by treating the gel with an alkaline (basic) solution. The gel is then neutralized prior to transfer
Southern Step #3
DNA transfer - the DNA in the gel is transferred to a membrane by one of three methods: 1. Capillary transfer 2. Vacuum transfer 3. Electrotransfer Membranes typically used are: 1. Nitrocellulose 2. Nylon (positively charged)
Southern Step #4
Crosslinking - the single-stranded DNA must be permanently attached to the membrane. This is done by crosslinking DNA to the membrane by one of the following methods:
- UV radiation
- Baking the membrane at high temp
Southern Step #5
Prehybridization - in order to ensure that the probe DNA will bind to the DNA on the membrane and not the membrane itself, the membrane is treated with hybridization solution that does not contain probe DNA
Southern Step #6
Hybridization - hybridization solution containing denatured (single-stranded) labeled probe DNA is added to the filter. The probe DNA should recognize and bind to complementary DNA
Southern Step #7
Washing - since excess probe DNA was used, it is necessary to wash off any probe DNA that did not hybridize to its complementary DNA. The stringency can be controlled by adjusting the following wash conditions:
- Temp
- Time
- Denaturant concentration (e.g., formamide, SDS)
- Na+ concentration (affects ionic strength)
Stringency
properties of hybridization conditions that affect the degree of base mismatches permitted between probe and target DNA
Southern Step #8
Exposure - different detection methods are necessary to determine where probe DNA bound to the membrane. Detection methods fall into two different categories:
- Direct = using radioactive phosphate (32P)
- Indirect = other methods of labeling and additional detection steps
Methods for labeling probe DNA
- End labeling by polynucleotide kinase - adds radioactive phosphate to 5’ end of DNA (no synthesis required)
- Random priming - random primers allow for nucleotide incorporation by DNA synthesis
- PCR - labeled nucleotides are incorporated into DNA by PCR
- Transcription in vitro - single stranded RNA is made that can be used as a probe
Radioactive nucleotides
32P is a radioactive isotope of phosphorous. To be used, 32P must be located in either the γ‐phosphate (end labeling) or the α-phosphate (random primer labeling, PCR, in vitro transcription). Detected by X-ray film (autoradiography)
3 major steps to label a primer by random priming
- Denature a DNA template (this is typically the DNA you wish to target by hybridization and anneal random primers)
- Add DNA polymerase (Klenow fragment), unlabeled dNTPs, and only one α-32P labeled dNTP (dATP, dCTP, dGTP, or dTTP)
- Once synthesis is complete (typically after about 1-2 hr), remove excess radioactive nucleotides and denature the probe DNA. This can now be used to hybridize to target DNA on a membrane
Modified nucleotide insertion by random priming
A nucleotide triphosphate (Digoxigen = DIG, biotin) can be incorporated into DNA in place of dTTP by any process that synthesizes DNA (e.g., random priming, PCR, in vitro transcription).
Digoxigenin (DIG)
a plant steroid, and there is a very-specific antibody available to this group
Biotin
A naturally-occurring vitamin that is bound tightly by the protein streptavidin
Digoxigenin detection
The probe DNA labeled with digoxigenin hybridizes to target DNA. The antibody to DIG can be conjugated with horseradish peroxidase (HRP) or alkaline phosphatase (AP). HRP or AP converts an appropriate substrate to compound that can be detected as a signal- identifying where the probe has bound
Biotin detection
The probe DNA labeled with biotin hybridizes to target DNA. Streptavidin is conjugated to HRP or AP. Streptavidin binds biotinylated probe DNA tightly, and HRP or AP converts an appropriate substrate to compound that can be detected as a signal
Uses for Southern hybridization
- Whether a specific DNA sequence is present or absent (sequence can be intragenic or extragenic)
- Is the sequence of interest present in different species (zoo blot)
- The amount (copy number) of a sequence within a genome
- The size of the restriction fragments where the sequence lies
Useful in forensic science and diagnosis of genetic diseases to indentify mutation, deletions, and gene rearrangements
DNA libraries
A tool that has been very useful in recombinant DNA studies
A DNA library is a large collection of DNA fragments that have been isolated, cloned, and can be propagated in an organism
2 major types of DNA libraries
- Genomic library - collection of entire genome of an organism
- cDNA library - collection of all genes that are expressed in a particular tissue type or under certain conditions
Genomic DNA library construction
- Human double stranded DNA cleaved with restriction nuclease
- Clone DNA fragments into vectors
- Introduce vectors into bacteria
Each bacterium contains a different region of genomic DNA. The entire collection of different bacteria containing different genomic DNA is called a library
Genomic DNA library uses
- Isolate genes from an organism
- Probe other organisms for similar genes/sequences
- Sequence entire genomes
Although the sequencing of whole genomes has made the first two uses somewhat less preferred, there are still many instances where genomic libraries are still preffered
Disadvantages of genomic DNA libraries
- Genes are often 10-100 kbp in length, and intact genes might not be cloned into one vector
- Genomic DNA in the region of a gene not only contains coding sequence (exons) but also non-coding sequence (introns) and regulatory regions
Solution: Only clone genes that are expressed by cloning the processed mRNA
Problem: Cannot clone RNA into DNA - the RNA must first be converted to double stranded DNA
mRNA processing
See slide 25
The resulting mRNA does not contain any introns and contains a poly A tail
Required steps to convert mRNA to ds DNA
- Oligo-dT primer is necessary to anneal to 3’ poly A tail of mRNA
- An enzymes called reverse transcriptase is then used to synthesize one stand of DNA from the mRNA template (this DNA is copy DNA or complementary DNA (cDNA))
- Once first DNA strand is synthesized, the RNA strand is degraded by treating with alkaline solution
- Terminal deoxynucleotide transferase can be used to add dGTPs to the 3’ end of the single stranded DNA
- Once additional G’s have been added, an oligo-dC primer can be used to prime DNA synthesis
- The other strand of DNA is synthesized, resulting in ds DNA
- The ds DNA is treated with a modifying enzyme, in this case EcoRI methylase (This will protect the DNA from digestion by EcoRI restriction enzyme
- Linker DNA containing an EcoRI restriction site is ligated onto the ends of the ds cDNA
- The cDNA can now be digested with EcoRI and cloned into a vector
- The collection of bacteria containing different cDNA vectors is called a cDNA library
cDNA uses
- Isolate expressed genes from an organism
- probe other tissues/organism cDNA libraries to examine gene expression in these cells
- Examine gene function by expressing cloned cDNA’s in cells (this use requires that the cDNA be expressed from a known promoter, since the cDNA itself does not contain any regulatory elements)
Genomic DNA vs cDNA
Slide 32, learn to draw
Colony hybridization
- Colonies on a plate are transferred to a membrane
- The transferred cells are then lysed and the DNA is denatured on the membrane
- The DNA is cross linked to the membrane
- The membrane is prehybridized and then hybridized with labeled single stranded probe DNA
- The excess probe is washed off
- The blot is developed to identify where the probe DNA bound
- Finally, the investigator can go back to the original bacterial plate and identify which colony contained their DNA of interest
