Chapter 16: Genome Analysis: DNA Typing, Genomics, and Beyond

1. Chapter 16: Genome Analysis: DNA Typing, Genomics, and Beyond

2. Some scientists said there was no reason to do it [The Human Genome Project] over 15 years. Why not do it over 25? One important reason is that if you did it over 25 years, most of the experienced scientists involved in it might be dead, at least mentally, by the time it was finished… Most people like to do things where they can see the results. James Watson, Genetics and Society (1993), p. 18.

3. 16.1 Introduction

4. Levels of genome analysis range from personal identification to comparative analysis of entire genomes.

5. 16.2 DNA typing

6. One of the most reliable and conclusive methods available for identification of an individual. • Technique developed by Alec Jeffrey’s and coworkers in 1985. • First called “DNA fingerprinting,” now called “DNA typing.”

7. Applications of DNA typing • Establish paternity and other family relationships.

8. Identify potential suspects whose DNA may match evidence left at a crime scene. • Exonerate persons wrongly accused of crimes. • Match organ donors with recipients in transplant programs. • Identify catastrophe victims.

9. Detect bacteria and other organisms that may pollute air, water, soil, and food. • Determine whether a clone is genetically identical to the donor nucleus. • Trace the source of different marijuana plants. • Identify endangered and protected species as an aid to wildlife officials.

10. DNA profiles of marijuana • DNA profiles generated by amplified fragment length polymorphism (AFLP). • Used to trace the source of marijuana samples to growers.

11. PCR amplification of restriction fragments to which adaptor oligomer sequences have been attached. • PCR primers recognize adaptors and bind to amplify different sized fluorescently-tagged DNA fragments. • Detected with a DNA sequencer.

12. Nonhuman DNA typing • DNA of protected whales found at Japanese markets • Incriminating pets • The case of the Rottweilers’ saliva. • The case of the hair from Snowball the cat.

13. DNA polymorphisms: the basis of DNA typing • Only about 0.1% of the human genome differs from one person to another. • With the exception of the human leukocyte antigen (HLA) region, genetic variation is relatively limited in coding DNA.

14. Less than 40% of the human genome is comprised of genes and gene-related sequences. • Intergenic DNA consists of unique or low copy number sequences and moderately to highly repetitive sequences.

15. The majority of DNA typing systems used in forensic casework are based on genetic loci with minisatellites or short tandem repeats (STRs). • Analyze multiple variable regions, called polymorphic markers.

16. The power of DNA evidence lies in statistics. • Aim to calculate the probability that only one person in a quadrillion (1015) could have the same profile of markers.

17. A variety of DNA technologies are used in forensic investigations: • Minisatellite analysis • PCR-based analysis • STR analysis • Mitochondrial DNA analysis • Y chromosome analysis • Random amplified polymorphic DNA (RAPD) analysis

18. Minisatellite analysis • Minisatellites are a special class of RFLP in which the variable lengths of the DNA fragments result from a change in the number, not the base sequence, of minisatellite repeats. • Also known as variable number tandem repeats (VNTRs).

19. Classic “DNA fingerprinting:” minisatellite analysis with a multilocus probe • Unique biological identifier for each individual. • Essentially constant for an individual, irrespective of the source of DNA. • Simple Mendelian pattern of inheritance.

20. Requires relatively large amounts of DNA. • Does not work well with degraded samples.

21. Minisatellite analysis with a single-locus probe • A single-locus probe allows the detection of a single minisatellite DNA locus on one chromosome. • To increase the sensitivity, 3-5 single-locus probes are mixed in a single-locus “cocktail.”

22. Polymerase chain reaction-based analysis • Sufficient DNA can be collected from saliva on a postage stamp or bones from skeletons. • Even highly degraded DNA can be amplified, as long as the target sequence is intact.

23. Short tandem repeat analysis • Currently the most widely used DNA typing procedure in forensic genetics. • The variability in STRs mainly occurs by slippage during DNA replication, rather than by unequal crossing-over.

24. Multiplex analysis of STRs • Simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. • The FBI uses a standard set of 13 specific STR regions for CODIS (The Combined DNA Index System).

25. Example: • 15 different STRs and a gender-specific marker amplified by PCR. • One primer in each pair is labeled with a fluorescent tag for 4 color detection. • Detect PCR amplification products using an automated sequencer. • Separated by size and detected by color after laser-induced excitation.

26. Mitochondrial DNA analysis • Every cell has hundreds of mitochondria with several hundred mtDNA molecules. • Older biological samples (e.g. strands of hair, solid bone, or teeth) often lack usable nuclear DNA but have abundant mtDNA. • mtDNA has been successfully isolated from fossil bones.

27. Analysis by PCR amplification and direct sequencing of two highly variable regions in the D loop region. • Can only identify a person’s maternal lineage.

28. Y chromosome analysis • Y chromosome-specific STRs. • Paternity testing of male offspring • Analyzing biological evidence in criminal casework involving multiple male contributors.

29. Randomly amplified polymorphic DNA (RAPD) analysis • No knowledge of an organism’s DNA sequence is required. • PCR primers consist of random sequences. • e.g. The case of the Palo Verde tree seed pods. • e.g. Differentiation between Bacillus species.

30. 16.3 Genomics, proteomics, and beyond

31. Whereas gene discovery once drove DNA sequencing, now the sequencing of entire genomes drives gene discovery.

32. What is bioinformatics? • Area of computer science devoted to collecting, organizing, and analyzing DNA and protein sequences and all the data being generated by genomics and proteomics labs.

33. Tools of bioinformatics: • Locate and align sequences. • Assemble consensus sequences. • Analyze properties of proteins. • Analyze sequence patterns to locate restriction sites, promoters, DNA binding domains, etc. • Phylogenetic analysis.

34. Basic local alignment search tool (BLAST). • The most commonly used genome tool. • Example: Search for all the predicted protein sequences that are related to a “query sequence.”

35. Genomics • The comprehensive study of whole sets of genes and their interactions rather than single genes. • Comparative analysis of genomes based on the availability of complete genome sequences.

36. Proteomics • The comprehensive study of the full set of proteins encoded by a genome—the “proteome.” • Protein biochemistry on a “high-throughput” scale.

37. The age of “omics” and systems biology • A whole set of related terms coined to describe the comparative study of databases. • e.g., transcriptomics, metabolomics, kinomics, glycomics, lipidomics • Interactomics: the study of macromolecular machines, mapping protein-protein interactions throughout a cell.

38. Systems biology aims to make sense of all the data arising from the study of biomolecular networks. • Uses both experimental and computational approaches to model these interactions. • “Attempts to piece together everything.”

39. 16.4 Whole genome sequencing

40. Major milestones in sequencing technology • Development of automated DNA sequencing. • Development of the BLAST algorithm. • Development of bacterial artificial chromosome (BAC) vectors.

41. Two main genome sequencing methods • Clone by clone genome assembly approach: • Used by the publicly funded international sequencing consortium for the human genome. • Whole-genome shotgun approach: • Used by the privately funded Celera Genomics Corporation for the human genome.

42. Clone by clone genome assembly approach • Restriction fragments of ~150 kb are cloned into BAC vectors. • A physical map of the genome is produced. • The BAC clones are broken up into smaller fragments, subcloned, and sequenced. • This places the sequences in order so they can be pieced together. • Time consuming, but precise.

43. Whole-genome shotgun approach • Plasmid clones with 2-10 kb inserts are prepared directly from fragmented genomic DNA. • Clones are randomly selected for sequencing. • Sequence is reassembled in order with the aid of a supercomputer. • More rapid, but often results in gaps in the sequence.

44. Rough drafts versus finished sequences • “Rough draft” of the human genome reported in 2001 by the publicly and privately funded groups. • “Finished” sequence reported in 2004. • More accurate and complete, but still contains some gaps.

45. Annotation of a sequenced genome is an in-depth analysis of all functional elements of the genome. • Much of the emphasis is on the gene content, with the aim of characterizing all of the genes and their functions.

46. Comparative analysis of genomes • Sequence and comparative analysis of nonmammalian genomes help to provide unique perspectives on the evolution of anatomy, physiology, development, and behavior.

47. Prior to the draft sequence, estimated that the human genome contained at least 100,000 genes. • Current estimate of 20,000 to 25,000 protein-coding genes came as a surprise.

48. What makes us uniquely human? • The answer lies somewhere within the 35 million single-nucleotide substitutions, 5 million small insertions and deletions, local rearrangements, and a chromosomal fusion that distinguish us from the chimpanzee (Pan troglodytes).

49. Comparative analysis of genomes: insights from pufferfish and chickens • Comparative genome analysis allows researchers to assess changes in gene structure and sequence that have occurred during evolution.

50. Homologous sequences share a common evolutionary ancestry. • Orthologs are genes in different species that are homologous because they are derived from a common ancestral gene. • Paralogs are two genes in a genome that are similar because they arose from a gene duplication.