RFLPs, AFLPs and Sanger sequencing

RFLP

  • Restriction fragment length polymorphism

  • Use restriction enzymes to cut DNA at short specific sequences

  • restriction enzymes: from bacteria, cleave foreign DNA (the bacterial DNA doesn’t get cut)

  • adding 1 or 2 restriction enzymes to purified dna turns a single piece of dna into multiple fragments

  • some individuals may only have one restriction site whereas others have two or three: the resulting fragments are different lengths

AFLP

  • amplified fragment length polymorphism

  • pcr-based

  • often used for plants

  • amplificaion of multiple fragments = diffrent-sized bands on a gel

  • pattern of bands depends on sequences immediately adjacent to adaptors and distance between restriction sites

Sanger sequencing

Transcription Translation

The sequence of base pairs in dna will determine sequence in the mRNA

three base pair sequences on mRNA are called codons

Uracil in RNA replaces thymine in DNA

Translation:

  • the sequence of bases in the codon determines the sequence of amino acids in the protein

  • redundancy in the genetic code means that many substitutions in DNA do not result in altered proteins (synonymous substitution)

    • ex. CUU, CUC, CUA, CUG all code for leucine

Junk DNA vs genes that code for products: Exons are transcribed, introns are not Most variations is in the introns

DNA sequences

  • Advantages: PCR-based

    • almost any tissue can be used

    • tiny amounts of tissue required

    • output is exact sequence of bases - can see lots of variation

  • Disadvantage

    • usually measures diversity within a single sequence

    • moderately expensive

Sequencing

Estimates of genetic variation: Haplotype diversity

  • e.g. mtDNA data

  • describes the numbers and frequencies of mtDNA haplotypes

  • Heterozygosity equivalent for haploid loci

  • Will often approach 1 if a high proportion if individuals have unique haplotypes

  • no assessment of the magnitude of the difference (just assesses whether they are different)

Nucleotide diversity (pi)

  • factors in both frequences and pairwise divergences of the different sequences

N = number of sequences in sample

pi and pj are frequency of sequences I and J in the sample and

piij is the proportion of sites that differ between sequences i and j