Exam II Genetics

2/19/24

• Next Generation Sequencing

  • Pyrosequencing - when a dNTP is incorporated into a growing DNA strand, two covalently linked phosphates (pyrophosphate, PPi) are released

    • pyrosequencing machines detect light every time these 2 phosphates are released and react chemically with enzymes to form ATP and give off life

    • dNTPs area one at a time, and removed if not incorporated into sequencing reaction

    • refer to textbook

• Site-specific Mutagenesis

  • Sit specific mutagenesis - in vitro targeting of specific cloned genes

    • Use PCR w/ mutant (M) primers

    • Heat products and allow to re-anneal (some A will bond with B)

    • DNA polymerase fill in gaps

    • Amplified again with primer 1 & 3 for full-length molecule with mutation

    • transform into cell - replaces wild type

  • Gene knockouts

    • How a gene affects human health

      • Clone human gene - then clone it in mouse

      • Site-specific mutagenesis of mouse gene

      • Inject mutant gene into mouse embryos — replaces one wild type gene in mouse cells

      • Screen baby mice for mutation — knockout mice heterozygotes (+/-)(One normal wild type copy & mutant copy)

      • Interbreeding of heterozygotes can produce homozygote knockouts (only mutant copies leftover)

  • Polymorphism

    • The quality or state of being able to assume different forms

      • In species — existence of several forms (shape, size, color) regardless of sex

  • DNA polymorphism: One of two or more alternative forms of an allele from a chromosomal locus resulting from differences in DNA sequences or numbers of tandem repeats-not necessarily a gene-can be anywhere

    • DNA marker: a known DNA sequence, often of DNA polymorphisms—best ones are unique in the genome, STS (sequence-tagged sites)

    • Classes of DNA polymorphisms: SNPs (single-nucleotide polymorphisms), STRs (Short-tandem repeats), and VNTRs (variable number of tandem repeats)

      • SNPs ~1 every 1 kbp (45% of genes are heterozygous) - 12 million SNPs identified in humans

      • Generally each gene is STS site

        • SNOS cause a single point mutation at a site—most common DNA polymorphism (95% of DNA variation)

        • Can be harmful if they occur in coding genes, or regulatory regions—detection is important

        • SNPS can sometimes affect restriction sites, thus causing Restriction Fragment Length Polymorphisms—restriction enzyme generated fragments of different lengths

        • RFLPs can be detected with Southern blot or PCR

  • Southern blotting

    • Analysis determines arrangement and location of restriction sites

      • Genomic DNA is digested with several restriction enzymes, produces many fragments

      • Fragments separated by size with gel electrophoresis

      • DNA fragments transferred to a membrane filter—arranged exactly as they are on the gel

      • a labeled probe (radioactive or chemiluminescence) binds to DNA interest

        • Southern blot: DNA

        • Northern blot: RNA

        • Western blot: Proteins

  • Oligonucleotide: Short piece of RNA or DNA

    • Used to find SNPs, as SNP do not attach to restriction sites

      • Make an oligonucleotide that matches normal allele perfectly

      • Isolate target DNA and mix with oligo-

  • Microarrays: Developed in 1990s, ordered grid of probe single-stranded DNA molecules of known sequence (with many SNP combinations) fixed to a microchip

    • Use unlabeled probes (fixed to substrate) and free target DNA from target organisms with colored dyes

    • Ex. cDNA targets have red or green des, so red or green spots on microarray indicate hybridized to indicate those probes

    • Yellow means both hybridized, other colors indicate degrees of hybridization

    • cDNA = DNA synthesized by reverse transcriptase from mRNA

  • Transcriptomics

    • Microarrays now largely replaced by transcriptomics

    • Study of the transcriptome: the complete set of RNA transcripts produced by the genome of a specific cell or organism at a specific time, or under a specific set of conditions

    • Transcriptome is dynamic/constantly changing

    • Entire transcriptome can be sequenced with next-gen sequencing (specifically RNA-seq. in this case) to understand what genes are turned off/on)

  • STRS, SSRs or microsatellites

    • Short tandem repeats or simple sequence repeats: 2-6 bp of DNA sequences repeated tandemly up to about 100x in the genome

      • CCTCCTCCTCCTCCTCCT

    • High rate of mutation because:

      • are in noncoding regions of the genome

      • often mismatched during replication, considered “junk DNA”

    • STRs make up 10% of mouse genome, lots of data

    • With PCR, often used in population-level genetics studies/identify relationship to individuals

    • PCR amplifies region with STRs — gel electrophoresis used to see fragment sizes

  • VNTRs or minisatellites

    • Variable-Number Tandem repeats

      • Similar to STRs but repeating unit is 7 - 20

      • Larger length of units = southern blot used more often than PCR

      • Can be multiple VNTR copies scattered around genome

      • VNTRs first use of DNA data to exonerate someone in a murder trial in 1988

  • DNA fingerprinting: Takes advantage of DNA polymorphisms to identify individuals for forensics

    • DNA from blood, RE isolated segment with marker, VNTR probes used with s. blot to obtain profile of everybody

    • Everybody is heterozygous—but baby matches one allele from mom and one from dad

• Gene therapy

  • Gene therapy is mostly for somatic cells to correct diseases present

    • Commonly for disorders with single mutation for which a normal clone is available

    • procedure — take out cells, introduce normal gene (transgene) with virus vectors into them (now a transgenic cell). replace in body.

    • sometimes normal gene replaces mutant in cell, ither tines is inserted elsewhere in the genome and must be expressed to work

  • Pedigree

    • Pedigree: pictorial representation of a family,

      • tracks inheritance of one or more characteristics

    • Proband: person for whom the pedigree is initiated

  • Autosomal Recessive traits:

    • Appear equally in male and females

    • skip gens

    • Consanguinity: Affected people on mating among closely related members

• Genetic testing

  • Genetic testing: identifies mutations in key genes responsible for disease

    • Does not always work:

      • Gene for disease not found in genome

      • Gene has many mutations

      • Presence of one gene doe4s not always result in disease

      • Many diseases are polygenic

  • Carrier detection (heterozygote screening): genetic testing of adults to calculate probability of baby having disease, uses blood

  • Cell-free (cfDNA) screening: Blood test of DNA fragments from placenta at 10 weeks—looks for chromosomal abnormalities and sex of baby

  • Prenatal diagnosis: Amniocentesis is done to perform genetic testing—now can be done on embryos before implantation

  • Newborn screening: testing newborn babies for genetic disease using blood sample