NUCLEIC ACID STRUCTURE II

Conditions favoring renaturation (also known as annealing or hybridization) include:

• lower temperature (allows hydrogen bonds to form).
• high salt (cations shield negatively charged phosphates, eliminating the repulsive force between strands).
• neutral pH (also allows hydrogen bonds to form).

Renaturation kinetics

• rapid cooling (e.g., rapid shift from 100 degrees C to 4 degrees C) leaves DNA essentially single-stranded.
• renaturation under stringent conditions (e.g., shift from 100 degrees C to temperature about 25 degrees C below the Tm) can result in perfect base-pairing of homogeneous DNA (i.e., reconstitution of original double-stranded DNA). Note: Mismatched sequences can be distinguished from perfectly matched sequences. The melting temperature is reduced 1 degree C for every 1% mismatch (for example, 1 mismatch out of a sequence of 25 is 4% mismatch and a 4 degree C drop in melting temperature).
• renaturation under stringent conditions using radioactively labeled nucleic acid probes allows detection of specific DNA and RNA molecules (Southern blot and Northern blot techniques respectively; see below and page 976 of the textbook).

Derivation of the integrated form of reassociation kinetics (click here)

Integrated form of the equation: C/C_o = 1/(1 + kC_ot) = fraction of single-stranded DNA

Note that when t = 0 (start of the reaction), C/C_o = 1 (all the DNA is single-stranded); when t is very large (infinite time of reaction), C/C_o = 0 (no single-stranded DNA); and when the reaction is half over (C/C_o = 1/2 when t = t_1/2), then C_ot_1/2 = 1/k = kinetic complexity.

Double-stranded DNA at A₂₆₀ = 1 is at a concentration of 50 µg/mL = 1.5 x 10^-4 moles nucleotide/liter; to achieve a C_ot = 1 with this concentration of DNA would require 6,666 seconds = 111 minutes = 1.85 hours.

Standard conditions: 0.12 M sodium phosphate buffer (= 0.18 M Na⁺), neutral pH, Tm -25 degrees C (typically about 70 degrees C), DNA fragmented to 300-600 base pairs.

The speed of the reaction can be increased at lower temperatures by high salt and organic solvents (for example, formamide). Increasing the temperature of the reaction increases the stringency; that is, the number of perfectly paired bases required for nucleating reassociation is only about 12 at 60 degrees C, but about 18 at 70 degrees C.

C_ot plots (Figure D2): The kinetic complexity is a molecular ruler.

Usually C/C_o is plotted against the logrithm of C_ot; but sometimes 1 - (C/C_o) = fraction of double-stranded DNA is plotted against the logrithm of C_ot (this just turns the plot upside-down).

For a homogeneous, unique DNA molecule, reassociation occurs over a range of 100 C_ot units (2 log C_ot units).

Reassociation of eukaryotic DNA usually occurs over a very broad range of C_ot values spanning up to eight orders of magnitude (8 log C_ot units). The reason for this is that eukaryotic DNA actually includes several kinetic components, each reassociating with its own characteristic kinetics (Figure D3).

Recent sequencing of the human genome has revealed astonishing detail about repeated sequences (Figure D7). Some regions of the genome are extraordinarily dense in repeats (Figure D8).

Figure D4 shows that there is good agreement between the kinetic complexity and the chemical complexity of eukaryotic genomes (with the exception of polyploid genomes which contain more than two copies of the chromosome set).

Do you know C_ot curves? Test yourself!

Southern blotting (page 976)

How can you find a specific piece of DNA in a mixture containing many DNA molecules? First we introduce a technique to separate DNA molecules by size (variations of gel electrophoresis can separate very large chromosomal sized DNA molecules as well as DNA molecules that differ by a single nucleotide). Moreover, gel electrophoresis can be done under conditions that preserve native DNA structure or denature DNA molecules. DNA has a negative charge and moves in an electric field toward the positive electrode (anode). The supporting matrix (gel) is usually agarose or polyacrylaminde. Figure D5 shows and example of a gel electrophoresis unit. We will soon discuss a method for specifically fragmenting DNA molecules using site-specific endonucleases (restriction endonucleases).

DNA molecules separated by size using gel electrophoresis can be transferred to a support matrix (for example, a nitrocellulose membrane) and hybridized to a radioactive "probe" (for example, ³²P-labeled DNA). This technique is called Southern blotting (Figure 25D.1)

Want to detect an average gene of about 10³ base pairs in a genome of roughly 10⁹ base pairs or 10³/10⁹ = 1/10⁶ = 1 part per million.

We can apply approximately 10 µg to 100 µg of total DNA per gel lane to be electrophoresed and blotted. We need about 100 cpm of radioactivity to detect a band on an autoradiogram. So, in the worst case, the 10³ base pair probe nucleic acid must have a specific activity of 100 cpm/(10 µg/10⁶) = 100 cpm/10^-5 µg = 10⁷ cpm/µg. Of course, if the probe were smaller than 10³ base pairs, say an oligonucleotide probe 20-30 bases long, then the specific activity would need to be more like 10⁹ cpm/µg.

Units of radioactivity:

1 Ci (Curie) = 3.7 x 10¹⁰ Bq (Becquerel) = 2.2 x 10¹² dpm.
1 Bq = 1 dps (disintegration per second) = 60 dpm.
Note: dpm does not necessarily equal cpm (counts per minute); this depends on the counting efficiency of the instrument.

New technologies using nucleic acid hybridization involve "gene chips" (Figure D6).

Do you know how to quantitate blotting experiments? Test yourself!