Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

DNA is restriction digested with restriction enzymes, individually (single-enzyme digest) and in combination (double digest). The restriction fragments are subjected to electrophoresis. The fragments are identified, either using UV absorbing dye or labeled probe. Double digest determines if fragment produced by one enzyme contains restriction sites for the other enzyme. Fragments are aligned by size. Enzyme 1: 8 kb, 6 kb, 3 kb or 3 kb, 6 kb, 8 kb 6 kb, 8 kb, 3 kb or 3 kb, 8 kb, 6 kb 8 kb, 3 kb, 6 kb or 6 kb, 3 kb, 8 kb Enzyme 2: 10 kb, 7 kb or 7 kb, 10 kb Double Digest: 3 kb fragment is split into 2 kb and 1 kb fragments. Restriction Mapping

Restriction Fragment Length Polymorphism (RFLP) Individuals can be identified according to RFLP genotype. RFLP locus could be linked to a gene, and thus be used as a diagnostic marker.

Use of Restriction Fragment Length Polymorphism I.Marker locus II.Diagnostic A.Medicine B.Forensics III.Assessment of Genetic Variation A.Within and between populations B.Within and between species

Restriction Mapping versus RFLP Mapping I.Restriction Mapping A.Based on physical analysis of DNA B.Based on restriction sites with no variation C.Mostly short-range (fine-scale) maps II.RFLP Mapping A.Based on recombination analysis of matings B.Based on restriction-site variation between homologous chromosomes C.Mostly longe-range (coarse-scale) maps

Other Useful Approaches 1.Single Nucleotide Polymorphisms (SNPs) Individuals differ in single nucleotides (every 11 to 300 bp in interval). 2.Simple-Sequence Length Polymorphisms (SSLPs) Very short repetitive DNA sequences are more polymorphic than RFLP sequences. These are also called Variable Number Tandem Repeats (VNTRs) - Minisatellite Markers - Microsatellite Markers

Simple-Sequence Length Polymorphisms 1.Minisatellite DNA These are 1 to 5 kb in length consisting of repeats 15 to 100 nucleotides in length and are identified by Southern analysis. 2.Microsatellite DNA These are tandem repeats of dinucleotides, commonly stretches of CA. These are identified by gel electrophoresis of PCR products. 5’ C A C A C A C A C A C A C A 3’ 3’ G T G T G T G T G T G T G T 5’

Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

Dideoxy DNA Sequencing Chain-terminating (dideoxy) nucleotide Use of dideoxy nucleotide in primer extension reaction will randomly arrest DNA synthesis.

Dideoxy DNA Sequencing 4 different reactions are conducted, each with a different type of dideoxy nucleotide. Fragments are separated by electrophoresis. Banding pattern is used to infer the base sequence of the original template strand.

Sequencing Gel Using RadioactivePrimerRemember… This is base sequence of synthesized strand. Migration 3’3’ 5’5’

Oligonucleotide primers can be tagged with fluorescent dyes instead of radioactive labels. A different colored dye can be used for each of the four reactions. Reading the DNA sequence from an automatic sequencer

In Search of Potential Genes Open reading frames (ORFs) are long stretches of DNA that start with ATG and end with a stop codon. A double- stranded DNA molecule has 6 possible reading frames, 3 for each strand.