Human Genomes, Disease Alleles/Genes, & Forensics

Classes 15-17

DNA polymorphisms

• sequence differences

• anonymous: don’t affect nature or amounts of any proteins (or ncRNA) in the body

• most don’t influence phenotype

• can serve as a DNA marker

Single nucleotide polymorphism (SNPs)

• particular base positions in the genome where alternative letters of the DNA alphabet distinguish some people from others

• most common type of genetic variant

• inherited co-dominantly (bi-allelic)

  • two alleles for each SNP locus

  • can be heterozygous or homozygous

• on average, occurs every 1000 (1kb) base pairs in any pairwise comparison

• don’t influence phenotype (are uncoded)

Deletion-insertion polymorphism (DIPs or InDels)

• short insertions or deletions of genetic material

• second most common form of genetic variations

• occur roughly every 10 kb

• can be 1-100s of base pairs

Simple sequence repeats (SSR or micro-satellites)

• loci that sequences of one or more bases that are repeated in tandem

• different alleles have different numbers of repeat units

• most common repeating units are one, two, or three-base sequences

  • meaning, either have one, two, or three types of nucleotide involved

• 3% of total DNA in genome, found once every 30kb

• in non-coding regions, have no effect

• in coding regions, remember trinucleotide repeat diseases (slipped mispairing)?

• highly polymorphic, often with over 10 alleles at a single locus

  • but since they have a low mutation rate (relatively stable), can serve as DNA markers

Copy number variants (CNVs)

• DNA length polymorphisms involving more than just a few nucleotides (like SSRs and DIPs)

• variable number of copies of large blocks of genetic material up to 1mb in length

• highly polymorphic, but stable

• 99% of alleles are inherited (not derived from a new mutation)

Pairwise comparison

comparing two genomes side by side

DNA fingerprint/profile

• genotype of 13 unlinked, polymorphic SSR loci

• unique to any one person (except identical twins)

• any one person only has two alleles for any given locus

• main point: it’s highly unlikely (statistically) that someone has the exact same alleleic combination for multiple loci by chance (would have to be related somehow)

Polymerase chain reaction (PCR)

• amplifies a target region of DNA

• requires only the smallest amounts of DNA

• uses two 16-30 base long oligonucleotides as primers

  • primers are the beginning and end of the target region

  • one oligonucleotide is complementary to one strand at one end while the other is complementary at the other end

  • primers are dyed to fluoresce different colors with the 13 SSRs

• put into gel electrophoresis

• can identify allelic variants for each locus based on the colors and sizes of the products

Haplotype blocks

• segments of DNA with particular sets of link SNP alleles that tend to travel together from one generation to another, because they are flanked by recombination hotspots

• DNA within blocks contain NO hotspots for crossing over

Genetic genealogy

• the basis of genetic analysis, that relatives share haplotype blocks

• more closely related = more haplotype blocks shared and the longer their uninterrupted shared DNA segments

Genetic relatedness

• estimated by the fraction of autosomal DNA shared

• each parent has two alleles of each SNP; each child inherits a random one of those two SNPs (from each parent)

  • means that the child will share half of their DNA with each parent, and with each sibling (on average)

Nucleic acid hybridization

• the ability of complementary single strands of DNA or RNA to come together to form double-stranded molecules

• need a perfect match between all nucleotides in primers and template

  • if there’s a mismatch, it’s less stable (so in experimentation, researchers can weed out the imperfect ones but taking advantage of the fact that only perfect matches can withstand a particular temperature)

Anonymous loci

• polymorphisms that don’t affect phenotype

• serve as molecular markers for specific regions of the genome

Allele-specific oligonucleotides (ASOs)

• short 20-40 base oligonucleotides that hybridize under the right conditions to only one of the two alleles at a SNP locus

• attach to solid support, like a chip of silicon

• turn DNA from genome into a probe by fragmentation, denaturing into single strands

DNA microarray

provide information about degrees of relatedness through the tracking of millions of polymorphs

Positional cloning

• strategy to identify defects causing hereditary diseases

• get information about the location of a disease gene by finding the polymorphic loci (known) that the mutation (unknown) is genetically linked with

• maps genes more precisely (as compared to gene mapping)

• limitation = phase problem

Uninformative cross (phase problem)

• don’t know the allele configuration

• cannot tell what allele a child got from what parent

• can’t perform linkage analysis

Informative cross

• CAN tell what allele child got from which parent

• at least one parent is doubly heterozygous

Allelic heterogeneity

• genetic diseases caused by a variety of different mutations in the SAME gene

• ex: Cystic Fibrosis

Compound/trans-heterozygotes

one copy of the chromosome has a different mutation than the other copy, BUT the disease still occurs because the two genome copies fail to complement

Locus heterogeneity

• genetic diseases caused by mutations in one of two or more DIFFERENT genes

• ex: deafness

Complex/quantitative traits

• many different genes influence the trait to different extents

• no single gene determines the trait

High-throughput/massively parallel sequencing

• allows for millions of individual DNA molecules to be sequenced simultaneously

• Different from Sanger in that…

  • DNA molecules are anchored in place when synthesized by DNA polymerase

  • timing of base addition is controlled to see what base is added

  • OH- group is protected and can be removed when dNTP needs to be reactive (does not stop synthesis permanently)

Whole-genome sequencing (in general)

• goal: to directly find DNA alteration that is the disease allele (as opposed to looking for a marker, then sequencing candidate genes)

• assumptions:

  • disease alleles are rare in the population

  • pedigree insight/knowledge of inheritance pattern