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The absorbance at 260 nm may be plotted against the temperature of a DNA

solution which will indicate that little denaturation occurs below approximately 70C,

but further increases in temperature result in a marked increase in the extent of

denaturation. Eventually a temperature is reached at which the sample is totally

denatured, or melted. The temperature at which 50% of the DNA is melted is termed

themelting temperatureorTm, and this depends on the nature of the DNA (Fig. 5.7). If

several different samples of DNA are melted, it is found that theTmis highest for those

DNAs which contain the highest proportion of cytosine and guanine, andTmcan

actually be used to estimate the percentage (CþG) in a DNA sample. This relationship

A

C

C

A

G C

P

5  end

OH

C G

G C

G U

A U

U A

U A

3  end (accepts phenylalanine)

Acceptor stem

G

G G

G

A

G

DC

DA

A

U

G

C

C

mG

m 2 G

CG

CG

AU

GmC

A

G

mG

C

U

mC UGUG

GACAC

G

U mA

T C

C

T loop

Variable loop

Anticodon stem

Anticodon loop

Anticodon

Gm A A

U Y

Cm A

A

T stem

A

D stem

D loop





Fig. 5.6Secondary structure of yeast tRNAPhe. A single strand of 76 ribonucleotides forms four double-stranded

‘stem’ regions by base-pairing between complementary sequences. The anticodon will base-pair with UUU or

UUC (both are codons for phenylalanine); phenylalanine is attached to the 3’ end by a specific aminoacyl tRNA

synthetase. Several ‘unusual’ bases are present: D, dihydrouridine; T, ribothymidine;ψ, pseudouridine; Y, very

highly modified, unlike any ‘normal’ base. mX indicates methylation of base X (m 2 X shows dimethylation); Xm

indicates methylation of ribose on the 2’ position.

144 Molecular biology, bioinformatics and basic techniques