Chapter 8

Question 1

How can one identify a specific gene sequence when the entire genomic sequence is present?

Answer 1

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

Question 2

Summary of Southern hybridization (8 steps)

Answer 2

1. Fractionate and separate DNA by gel electrophoresis
2. Denature DNA by soaking gel in alkaline solution followed by neutralization solution.
3. Transfer the DNA from the gel to a membrane
4. Crosslink the DNA to the membrane
5. Prehybridize the membrane with a solution that does not contain probe DNA.
6. Hybridize membrane with a solution containing denatured labeled DNA probe
7. Wash the membrane
8. Develop the membrane

Question 3

Southern Step #1

Answer 3

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

Question 4

Southern Step #2

Answer 4

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

Question 5

Southern Step #3

Answer 5

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)

Question 6

Southern Step #4

Answer 6

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:

1. UV radiation
2. Baking the membrane at high temp

Question 7

Southern Step #5

Answer 7

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

Question 8

Southern Step #6

Answer 8

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

Question 9

Southern Step #7

Answer 9

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:

1. Temp
2. Time
3. Denaturant concentration (e.g., formamide, SDS)
4. Na+ concentration (affects ionic strength)

Question 10

Stringency

Answer 10

properties of hybridization conditions that affect the degree of base mismatches permitted between probe and target DNA

Question 11

Southern Step #8

Answer 11

Exposure - different detection methods are necessary to determine where probe DNA bound to the membrane. Detection methods fall into two different categories:

1. Direct = using radioactive phosphate (32P)
2. Indirect = other methods of labeling and additional detection steps

Question 12

Methods for labeling probe DNA

Answer 12

1. End labeling by polynucleotide kinase - adds radioactive phosphate to 5’ end of DNA (no synthesis required)
2. Random priming - random primers allow for nucleotide incorporation by DNA synthesis
3. PCR - labeled nucleotides are incorporated into DNA by PCR
4. Transcription in vitro - single stranded RNA is made that can be used as a probe

Question 13

Radioactive nucleotides

Answer 13

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)

Question 14

3 major steps to label a primer by random priming

Answer 14

1. Denature a DNA template (this is typically the DNA you wish to target by hybridization and anneal random primers)
2. Add DNA polymerase (Klenow fragment), unlabeled dNTPs, and only one α-32P labeled dNTP (dATP, dCTP, dGTP, or dTTP)
3. 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

Question 15

Modified nucleotide insertion by random priming

Answer 15

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).

Question 16

Digoxigenin (DIG)

Answer 16

a plant steroid, and there is a very-specific antibody available to this group

Question 17

Biotin

Answer 17

A naturally-occurring vitamin that is bound tightly by the protein streptavidin

Question 18

Digoxigenin detection

Answer 18

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

Question 19

Biotin detection

Answer 19

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

Question 20

Uses for Southern hybridization

Answer 20

1. Whether a specific DNA sequence is present or absent (sequence can be intragenic or extragenic)
2. Is the sequence of interest present in different species (zoo blot)
3. The amount (copy number) of a sequence within a genome
4. 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

Question 21

DNA libraries

Answer 21

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

Question 22

2 major types of DNA libraries

Answer 22

1. Genomic library - collection of entire genome of an organism
2. cDNA library - collection of all genes that are expressed in a particular tissue type or under certain conditions

Question 23

Genomic DNA library construction

Answer 23

1. Human double stranded DNA cleaved with restriction nuclease
2. Clone DNA fragments into vectors
3. 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

Question 24

Genomic DNA library uses

Answer 24

1. Isolate genes from an organism
2. Probe other organisms for similar genes/sequences
3. 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

Question 25

Disadvantages of genomic DNA libraries

Answer 25

1. Genes are often 10-100 kbp in length, and intact genes might not be cloned into one vector
2. 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

Question 26

mRNA processing

Answer 26

See slide 25

The resulting mRNA does not contain any introns and contains a poly A tail

Question 27

Required steps to convert mRNA to ds DNA

Answer 27

1. Oligo-dT primer is necessary to anneal to 3’ poly A tail of mRNA
2. 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))
3. Once first DNA strand is synthesized, the RNA strand is degraded by treating with alkaline solution
4. Terminal deoxynucleotide transferase can be used to add dGTPs to the 3’ end of the single stranded DNA
5. Once additional G’s have been added, an oligo-dC primer can be used to prime DNA synthesis
6. The other strand of DNA is synthesized, resulting in ds DNA
7. 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
8. Linker DNA containing an EcoRI restriction site is ligated onto the ends of the ds cDNA
9. The cDNA can now be digested with EcoRI and cloned into a vector
10. The collection of bacteria containing different cDNA vectors is called a cDNA library

Question 28

cDNA uses

Answer 28

1. Isolate expressed genes from an organism
2. probe other tissues/organism cDNA libraries to examine gene expression in these cells
3. 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)

Question 29

Genomic DNA vs cDNA

Answer 29

Slide 32, learn to draw

Question 30

Colony hybridization

Answer 30

1. Colonies on a plate are transferred to a membrane
2. The transferred cells are then lysed and the DNA is denatured on the membrane
3. The DNA is cross linked to the membrane
4. The membrane is prehybridized and then hybridized with labeled single stranded probe DNA
5. The excess probe is washed off
6. The blot is developed to identify where the probe DNA bound
7. Finally, the investigator can go back to the original bacterial plate and identify which colony contained their DNA of interest