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7. Studying genetic diversity in species.

Dna sequencing

The new technology allows direct sequencing of DNA fragments rather than trying to figure out the gene order, DNA mutations and new genes by traditional methods such as RFLP analysis, chromosomal walking or even transduction and conjugation experiments in bacteria. DNA sequencing has now reached the automated stage and is routinely used in many laboratories even for HLA typing. In automated sequencing, a single sequencing reaction is carried out in which the four ddNTPs are labeled with differently colored dyes. At the end of the reaction, the mixture is run in a polyacrylamide gel, and the colored chains are detected as they migrate through the gel. The detection system identifies the terminal base from the wavelength of the fluorescence emitted upon excitation by a laser. The DNA polymerase used in a sequencing reaction is usually part of the E.coli polymerase known as the Klenow fragment or a genetically modified DNA polymerase from the phage T7 (Sequenase). The usual Taq DNA polymerase can also be used for this purpose.

Lecture 16. gene and dna analysis


As we know the knowledge of gene structure is extremely important for gene manipulation as well as for understanding basic principles of life. The common structure of a gene is shown below.

Sequence Element Function
Promoter To target RNA polymerase to DNA and to start transcription of a mRNA copy of the gene sequence.
Transcription terminator To instruct RNA polymerase to stop transcription.
Shine-Dalgarno sequenceand translation start S-D sequence in mRNA will load ribosomes to begin translation. Translation almost always begins at an AUG codon in the mRNA (an ATG in the DNA becomes an AUG in the mRNA copy). Synthesis of the protein thus begins with a methionine.
Coding Sequence Once translation starts, the coding sequence is translated by the ribosome along with tRNAs which read three bases at a time in linear sequence. Amino acids will be incorporated into the growing polypeptide chain according to the genetic code.
Translation Stop When one of the three stop codons [UAG (amber), UAA (ochre), or UGA] is encountered during translation, the polypeptide will be released from the ribosome.

Example: A gene coding sequence that is 1,200 nucleotide base pairs in length (including 1200 the ATG but not including the stop codon) will specify the sequence of a protein/= 3400 amino acids long. Since the average molecular weight of an amino acid is 110 da, this gene encodes a protein of about 44 kd, the size of an average protein.

Classically, genes are identified by their function. That is, the existence of the gene is recognized because of mutations in the gene that give an observable phenotypic change.

Historically, many genes have been discovered because of their effects on phenotype. Now, in the era of genomic sequencing, many genes of no known function can be detected by looking for patterns in DNA sequences. The simplest method which works for bacterial and phage genes (but not for most eukaryotic genes as we will see later) is to look for stretches of sequence that lack stop codons. These are known as open reading frames or ORFs. This works because a random sequence should contain an average of one stop codon in every 21 codons. Thus, the probability of a random occurrence of even a short open reading frame of say 100 codons without a stop codon is very small (61/ 64)100 = 8.2 x 10-3

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Source:  OpenStax, Genetics. OpenStax CNX. Jul 29, 2009 Download for free at http://cnx.org/content/col10782/1.1
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