Biology 471 - Molecular Biology and Biotechnology - Genetic fingerprinting

DNA Fingerprinting

          - a method to rapidly screen for genetic identity

           a) harbour specific alleles for specific genes

           b) family identification

           c) individual identification

- genes evolve independently in separated lineages

          - amount of sequence divergence (genetic distance) in a population depends on several things

- time since all copies of the gene can be traced to a common "ancestor" (Image)

          - may be more recent than the origin of the species

          - e.g. mitochondrial "Eve"

- rate of mutation of the gene

          - mtDNA vs. rDNA

- effective population size

          - decreased by

                     - population bottleneck

                     - non-random mating

- crossing over between chromosomes

          - tends to be inter-genic

- insertion/deletion of bases, base substitutions

          - restriction fragment differences

          - the more differences in restriction fragments, the greater the divergence in total sequence

- PCR or Southern blots

- single- or multi-locus probes

- single locus probes used in medical diagnoses (presence/absence of a specific gene allele)

          - diseases that are caused by a mutation in a single gene

          - Huntington's, Tay-Sachs, thallasaemia, cystic fibrosis

          - caution!

                     - vast majority of diseases are influenced by more than one gene!

                     - variable penetrance (BRCA1)

          - must first find a restriction site that is polymorphic, with one state (i.e. present or absent) strongly corresponding to the mutant state

                     - called a "linked marker"

                     - Sickle-cell anaemia

                                - DdeI polymorphism in the -globin gene

                                - due to a single base substitution, replace valine with glutamic acid

                                - use portion of cloned beta-globin gene

                     - many important diseases not as clear cut (BRCA1)

                                - many mutations

- forensic uses of single locus probes

          - single probe can give identical result for more than one person, even if several enzymes are used

- Variable number tandem repeats (VNTR's) (Image)

          - 15-100 bp in length, each unit

          - cause restriction fragment length variation without changes to restriction sites

          - "mini-satellites"

          - rapid mutation rate

- no matter how sensitive the probe, there is always a chance that the DNA profile can match more than one person

          - DNA evidence alone can acquit a suspect (Guy-Paul Morin)

          - DNA evidence alone cannot convict a suspect (O.J. Simpson)

- non-forensic identification

          - use several probes to different parts of the genome

          - much more randomized due to independent segregation of chromosomes

- multi-locus probes used for family studies (Image)

          - target highly repetitive DNA in the eukaryotic genome

          -"satellite DNA": has a different density than rest of genome

          - humans have Alu sequences, contain an AluI restriction site

                     - several places in the genome, different chromosomes

                     - blot restricted DNA, and probe with multi-locus probe

                     - will hybridize to many different fragments

                     - many will be too light to see, some will be stronger

                     - parent/child or siblings should share 1/2 of probed sites

- PCR is used when the amount of available DNA is too small

          - PCR/RFLP (single-locus)

          - RAPD (multi-)

          - AFLP (multi-)

Ancient DNA

          - DNA can survive for a long time under the right conditions

                     - dessication

          - fragments of DNA that are millions of years old can be recovered

          - NOT entire genes, or significant portions of a genome (good-bye Jurassic Park)

          - genomes that are thousands of years old may be able to be recovered

                      - wolly mammoth

Microarray chip (Image)

          - aka GeneChip^® probe array

          - developed by Affymetrix Corp.

- look for the expression of many specific genes in a tissue

          - made possible by the burdgeoning field of genomics

- all of the genes in an organisms genome

- all of the genes expressed in a tissue

          - e.g. the Drosophila genome array

                     - D. melanogaster genome recently worked out

                      - have sequence details on 13,500 genes

- for each gene an oligonucleotide probe is designed

- each one synthesized onto one spot of the chip, one nucleotide at a time

- total mRNA from a tissue is taken, and labelled with a tagged nucleotide (like automated sequencing)

- the chip is then flooded with the tagged mRNA

- if a mRNA is homologous to a probe on the array, it will anneal

          - each spot with the sequence for a gene that is expressed with have the tag present

- the chip is "read" with a scanning laser beam

- compare organisms at different developmental stages or under different physiological challenges

- compare gene expression in genetically modified organisms!