Exam II Genetics

UTEP BIOL 3320 Greenbaum

Pyrosequencing

A method in Next Generation Sequencing where the release of two covalently linked phosphates (pyrophosphate, PPi) upon incorporation of a dNTP into a DNA strand is detected as light signals.

Site-specific Mutagenesis

In-vitro targeting of specific cloned genes using PCR with mutant primers, followed by DNA polymerase filling in gaps and re-annealing to introduce mutations.

Gene Knockouts

Technique to study gene function by replacing a wild type gene with a mutant gene in cells, leading to knockout mice with homozygote mutations.

Polymorphism

The existence of different forms in species, including DNA polymorphisms like SNPs, STRs, and VNTRs, which are variations in DNA sequences or numbers of tandem repeats.

Southern Blotting

A technique to determine the arrangement and location of restriction sites in DNA by digesting genomic DNA, separating fragments by size, transferring to a membrane, and using labeled probes to detect specific sequences.

Transcriptomics

Study of the transcriptome, the complete set of RNA transcripts produced by an organism, often analyzed using next-gen sequencing like RNA-seq to understand gene expression.

STRs, SSRs, or Microsatellites

Short tandem repeats or simple sequence repeats, used in population genetics studies due to their high mutation rate and presence in noncoding regions of the genome.

VNTRs or Minisatellites

Variable-Number Tandem Repeats with repeating units of 7-20 bp, often used in DNA fingerprinting for forensic identification purposes.

Gene Therapy

A method to correct genetic disorders by introducing normal genes into somatic cells using virus vectors, aiming to replace or supplement defective genes.

Pedigree

A visual representation of a family's inheritance patterns, including autosomal recessive traits and consanguinity, used to track the transmission of genetic characteristics.

Genetic Testing

Identifying mutations in key genes responsible for diseases, including carrier detection, cell-free screening, prenatal diagnosis, and newborn screening for genetic disorders.

Homologous

A chromosome pair (one from each parent) that contain some genes and pair during meiosis

Autosomes

Non-sex chromosomes

Metacentric

Centromere centered

Submetacentric

Centromere positioned so one arm is longer than the other

Acrocentric

centromere close to end so that p-arm is tiny and hard to observe

Telocentric

Centromere is on very end, so chromosome has only one arm

Holocentric

entire chromosome acts as centromere

In somatic cells, alternation between interphase and mitosis is controlled by

regulatory molecules (cyclins & cyclin-dependent kinases)

Interphase - mitosis

DNA/chromosome replication

Prophase - mitosis

chromosomes thicken, spindle fibers form, kinetochore binds to centromere

Mutation

The process of alteration of a gene or chromosome; the end product of this process

Halotype

a particular DNA sequence that differs by one or more mutations from homologous sequences

Genotype

Unique genetic makeup of an individual, usually in relation to a specific gene

Richard Goldschmidt

Geneticist; one of the first to integrate genetics, development and evolution

Proponent of mutationist & “hopeful monster” theories

Mutationist theory

New species can arise by mutations

Polygeny

Many loci affect the same trait; means mutation is limited

Loci/locus

The exact position for a gene on a chromosome

Limits of mutation

Limited to alternation of preexisting traits or developmental pathways

Charles Darwin & speciation

Heritable adaptive variation is due to random mutations, differential survival due to natural selection

Heritable variation

Characteristics of an individual that are transmitted from generation to generation

Phenotype

Observable characteristics of an organism produced from interaction of genotype & environment

2 individuals with the same genotype can have..?

The same phenotype

2 individuals of the same genotype can have..?

The same phenotype

Dizygotic twins (fraternal; two fertilized eggs by two different sperm) can show

The same phenotype with different genotype

Average human mutation rate

1 in 30 mil bp (diploid genome 6.6 × 10^9 bp)

How mutation happens

Point mutation (deletion, insertion, or substitution of a bp)

Chromosomal mutation (chromosomal insertion, deletion, or rearrangement)

Point mutations result in _______ only if they occur in coding region of genes or regulating sequences

Gene mutations

Somatic mutations

Occur in somatic cells and only affect the individual in which the mutation arises

Germ-line mutations

Alter gametes and passed onto the next generation

Transition mutations occur at a higher frequency than..?

Than transversion mutations

Base pair substitutions (mutations at one nucleotide) can result in

Synonymous/silent mutations - no effect on amino acid translation

Non-synonymous mutations - change amino acid translation

Neutral mutation

All synonymous and some non-synonymous mutations can result in NO effect on phenotype or major effects; type of missense mutation that results in a different amino acid but functionally stays the same

Missense mutation

Change from one amino acid to another

Nonsense mutations

Change from one amino acid to a stop codon (UAA, UAG, UGA)

Silent mutations

Do not result in amino acid change

Tautomers

Different chemical states of nitrogenous bases

Keto form

Normal state of tautomers

Enol form - tautomers

Hydroxyl group (OH) affixed to a carbon with a double bond

Imino form - tautomers

Double bond migrates and moves position of hydrogen bonds

Spontaneous mutations: no agent

DNA replication errors & molecular changes

DNA replication errors examples

base substitution (tautomers & specific transitions)

Deletions & additions

Examples of molecular change mutations

Depurination (1 purine removed, polymerase stalls at missing base)

Deamination (amino group removed from base)

Chemically and radiation induced mutations

Ionizing and UV light

Base analogs

Base modifiers

Intercalators

Carcinogens

Site-specific mutatgenesis

Specific mutation within a gene; mutant gene introduced in normal cell to observe effects on phenotype

Ionizing and UV lights result in what kind of mutations?

Point mutations + thymine dimers

Base analogs high frequency tautomers & base modifiers result in

Specific transitions

Intercalators result in what kind of mutations?

Frameshifts in genes as a result of deletions/additions

Carcinogens

Mutagens that result in cancer

Thymine dimers (T^T)

Disrupts base pairing A-T and causes a bulge in DNA, disturbing DNA replication at the bulge, leading to cell death (skin cancer)

Base excision repair

DNA polymerase reverses the affect mutations have on DNA damage; removes mutated base and replaces it with correct one

Base excision repair steps

1. Uracil-DNA glycosylase: cleaves bond between sugar and base

2. Endonuclease cleavage: cuts away baseless sugar

3. DNA Polymerase activity: inserts correct base

4. Ligase activity: bonds new base to others in DNA strand

Nucleotide excision repair

Proteins can correct thymine dimers or other serious DNA distortions

UvrAB helicase and DNA pol I work to cut and replace damage, DNA ligase joins segments

Homozygous

Two alleles of the same trait (YY or yy)

Heterozygous

Two alleles of traits are different (Yy)

Transposable elements

Normal components of prokaryote and eukaryote genomes that encode proteins non-homologous recombination

Non-homologous recombination

the process of proteins jumping from one part of the genome to another; copying the gene and inserting it elsewhere

When transposable elements are inserted into a regulatory region, what does it do?

Enhances or destroys the regulatory region

Transposable elements can jump anyplace randomly with the effects of:

Null mutation, neutral effects non-coding insertion, chromosome mutations

what is the most likely reason as to why transposable elements are rare?

Frequent transposable element activity might kill the cell

Barabara McClintock

Discovered transposons in corn & won the 1983 Nobel Prize for it

Ac transposable element is

Autonomous

Ds is nonautonomous, meanjng

It needs autonomous transposable elements to activate it

Mendel’s 1st law

Law of segregation; two forms of gene (alleles) separate from each other during meiosis and gamete formation

Mendel’s 2nd law

law of Independent assortment; genes for different traits have independent hereditary paths

According to Mendel’s 1st law, progeny arise from..?

Random combination of two alleles—one from eggs and one from pollen

Monohybrid cross

A cross between two pure-breeding strains of peas that differ in a single trait

Reciprocal cross

Reversing the sexes involved in a cross produces the same result in peas

Diploid organisms produce haploid gametes, only one allele is given to each gamete. What is the possibility of getting a dominant (Y) or recessive (y) allele?

1/2

CRISPR

cRNA evolved to cut up foreign viral and plasmid DNA

CRISPR-Cas9 system derived from streptococcus pyogenes (strep throat) —> sgRNA guide; pairs with any DNA sequence & works with DNA repair mechanisms to disable genes/insert donor DNA

Cloning

The process of creating copies of DNA fragments and the protein products of DNA

Cloning tools

DNA from organism of interest

Restriction enzyme (endonucleases)

DNA ligase (joins recombinant DNA molecules)

Cloning vector (vehicle for foreign DNA replication)

Host microorganism (bacteria)

Restriction sites

Specific DNA sequences, often palindromes, that are recognized by restriction enzymes which cut out viral DNA

Restriction enzyme cutting - blunt ends

Restriction enzyme cuts both strands of DNA between two same base pairs

Restricion enzyme cutting - sticky (overhanging) ends

Restriction enzymes make staggered cuts in DNA

Why are sticky ends useful?

Recombinant DNA has the gene of interest, can combine by complementary base pairing to any other DNA fragment with a sticky end

Plasmid vectors

Ensures abundance of copies of recombinant DNA; include circular DBA and replicate separately from bacterial chromosome

Plasmid vectors are composed of

An ori sequence, needed for independent replication in bacteria

A selectable marker to identify bacteria

At least one restriction site for recombinant DNA insertion

pUC19 vector

100 copies per cell

ampR selectable marker

Multiple cloning site with several unique restriction sites (Located in lacZ+ coding gene)

When a plasmid inserts DNA into the vector and the appearance is white, what does this mean?

The DNA is live

PCR

invented by Kary Mullis, used to make and amplify many copies of any DNA template

Why is PCR important?

It is used for cloning for DNA sequencing and can help piece together DNA-based phylogeny, as well as diagnosis of genetic disease and genetic fingerprinting

Components of PCR

Buffer (Mg++)

Template DNA

2 primers to amplify DNA fragment

dNTPs

Taq DNA polymerase

What are the disadvantages of PCR?

The inability for Taq to proofread, if mismatch errors occur early in the PCR process, the mistake is magnified millions of times

Complementation test

Determines whether mutants affect same or different genes

If individuals with one of 2 alternate mutants are crossed, offspring’s phenotype tells us if mutants on different genes

Compound heterozygotes

Have two different alleles at a locus that result in a recessive phenotype

Example of the complementation test

Homozygous recessive mutant (e) gene gives black, mutant phenotype

If another individual has homozygous recessive mutant (b) genes, it has black mutant phenotype

Mutants are on different genes, complementing each other

A diploid individual cannot have more than..?

Two alleles of a gene (heterozygote)

Complementation test can determine if mutant phenotype is due to

different alleles or different genes