Genetics Unit 4

Mutations

• inherited change in DNA sequence

• source of genetic variation, useful for genetic dissection, responsible for disease

• caused by internal/normal and external/environmental factors (spontaneous or induced), chemicals can induce mutations

Somatic Mutations

• occur in nonreproductive cells

• passed to new cells through mitosis, creating a clone of cells having the mutant gene

• mitosis (nonsexual reproduction)

• numerous (many somatic cells)

Germ-line Mutations

• occur in cells that lead to gametes

• meiosis allows mutations to be passed to half members of next generation who carry mutations in cells

• meiosis (sexual reproduction)

Base Substitution

• transition (purine → different purine/pyrimidine → different pyrimidine

• transversion (purine replaced with pyrimidine/pyrimidine replaced with purine

Insertions/Deletions

• leads to even number of nucleotides (2 or 4) and changes codon

• may lead to frameshift mutation

• alter all of the codon after mutation

• can be in-frame if they don’t affect reading frame

Expanding Nucleotide Repeats

1. DNA molecule has eight copies of a codon repeat

2. two strands seperate and replicate

3. hairpin forms on newly synthesized strands causing parts of template strand to be replicated 2x (increasing number of repeats on newly synthesized strand

4. 2 strands of new DNA molecule separate and strand with extra codon copies serves as template for replicatio

5. resulting DNA molecule contains 5 additional copies of codon repeat

• involved in asl, fragile-x syndrome and 50 other diseases

Missense Mutation

• effect of mutation

• new codon encodes a different amino acid, there is a change in amino acid sequence

Nonsense Mutation

• effect of mutation

• new codon is a stop codon, premature termination of translation

Silent Mutation

• effect of mutation

• new codon encodes the same amino acid, no change in amino acid sequence

Suppressor Mutation

hides effect of another mutation

1. forward mutation changes wild type into mutant phenotype

2. reverse mutation restores the wild-type gene and phenotype

3. suppressor mutation occurs at site different from that of original mutation and produces and individual that has both original and suppressor mutation and wild-type phenotypye

• intragenetic: occur in gene containing mutation

• intergenetic: occurs in gene other than one with original mutation

Mutation Rates

frequenct of a wild-type allele at a locus changes into a mutant allele

Mispairing

• cause of mutation udring replication

• tautomeric shifts (position of protons in DNA bases change)

• wobble

Incorporated Errors

base substitution causes a mispaired base to be incorporated into a newly synthesized nucleotide chain

• wobble base pairing may lead to replicated error

1. DNA strands separate for replication

2. thymine on the original template strand base pairs with guanine through wobble (leads to incorporated error)

3. at next round of replication, the guanine nucleotide pairs with cytosine, leading to a transition mutation

Strand Slippage

1. newly synthesized strand loops out results in addition of one nucleotide on new strand

2. template strand loops out resulting in omission of one nucleotide on new strand

Unequal Crossing Over

if homologous chromosomes misalign during crossing over one crossover product contains an insertion and other has a deletion

Depurination

loss of purine base from a nucleotide may lead to base substitution

1. apurinic site connot provide a template for a complementary base on the newly synthesized strand

2. nucleotide with the incorrect base is incorporated into the newly synthesized strand

3. at next round of replication this incorrectly incorporated base will be used as a template leading to a permanent mutation

4. a nucleotide is incorporated into the newly synthesized strand opposite the apurinic site

Deamination

loss of amino group from a nucleotide can alter the pairing properties of a base

5-methylcytosine → thymine is common and a mutation hotspot in humans caused by base deamination

Base Analogs

chemicals with structures similar to those of any of the four strandard nitrogenous bases of DNA

ex. 5-bromouracil can become incorporated into DNA in place of thymine, producing an incorporation error and could lead to (can lead to permanent mutation)

Ionizing Radiation

capable of penetrating tissues and damaging DNA beacause of their high energy

• radiation dislodges electrons from atoms they encounter, alter structures of bases and breaks bonds

UV Radiation

• less energy, doesn’t dislodge electrons

• still highly mutagenic

Pyrimidine Dimers

created by pyrimidines adjacent to bases on the same strand that absorb UV light

• create chemical bonds

• form lesions that distort configuration DNA and block replication

Transposable Elements

DNA sequences that can move about in genome

• often cause of mutationsinsert themselves at many different locations in the genome

• insert/disrupt or promote chromosome rearrangement (deletion, duplication, inversions)

• 45% human genome comprises sequences relatde to transposable elements. (Alu = common)

• affect genome size variation based on number of copies of elements

• recombination causes gene duplication/chromosome rearrangement

• can move DNA sequences to new sites

Flanking Direct Repeats

generated when a transposable element inserts into DNA

• repeats flanking direct repeats to fill in cut out DNA

Transposition

movement of a transposable element from one location to another

1. staggered breaks made in target DNA

2. transposable element is joined to single-stranded ends of target DNA

3. DNA is replicated at single-strand gaps

• insertion of transposable element can cause mutations

Transposable Enzyme

often encoded by the transposable element

• used to make staggard breaks in DNA and to integrate transposable element into a new site

DNA transposons

• class II transposable elements

• transpose as DNA

Retrotransposons

• transpose through an RNA intermediate

• RNA trnscribed from transposable element (DNA) and coped back into DNA by reverse transciptase

• use replicative transposition only

Nonreplicative Transposition

• cut and pase transposition

• trnasposable element is excised from old site and inserted into new site without an increase in number of copies

Replicative Transposition

• copy and paste transposition

• new copy of transposable element is introduced at a new site while old copy remains behind at original site

Damaging Agents

• assult of DNA by radiation, chemical mutagens and spontaneously arising changes

• rate of mutation is low due to DNA repair efficiency

DNA-repair mechanisms

• most require nucleotide strands of DNA (template strands specify base sequence)

• many types of dNA damage can be corrected by more than one repair system

• diseases can be causes (especially cancer) due to faulty DNA repair

Mismatch Repair

corrects incorrectly inserted nucleotides that escape proofreading by DNA polymerase during replication

Direct Repair

restores original structures (doesn’t replace nucleotides

ex. direct repair corrects O6- methylguanine by sending an enzyme to remove the methyl group

Base-Excision Repair

modified base is excised and entire nucleotide is replaced

• catalyzed by DNA glycosylase (recognized and removes specific type of modified base)

Nucleotide-Excision Repair

removes bulky DNA lesions that distort the double helix

• 2 strands of DNA are separated and section of DNA (containing distortion) is removed, resulting gap is filled in by dNA polymerase and DNA ligase seals gap in sugar-phosphate linkage

Hutchinson-Gliford Syndrom

• causes premature aging

• CRISPR-Cas genome editing can fix genetic mutation

Restriction Enzymes

recognize specific nucleotide sequences in DNA and make double-stranded cuts at sequences to produce blunt/cohesive ends

• produced by bacteria to defend against viruses

• used in recombinant DNA technology

CRISPR-Cas Genome Editing

• modifies genome by incorporating invader DNA to provide memory which is then used to make defenses against it

• requires PAM sequence (short and week sequences with target) to function

• binds pam, unwinds DNA, pairs sgRNA with sequence, cleaves DNA

• either nonhomologous end joining (ends joined without template) or homologous directed repair (donor dsDNA provided by researcher_

• works in many organisms

• used in recombinant DNA technology

• sometimes targets cleavage of DNA that you don’t want cleaved

Gel Electrophoresis

• separates molecuels based on size and electrical charge

• probe (DNA/RNA with base sequence complementary to interest gene) can locate individual genes/sequence among DNA fragments

Southern Blotting

• isolates DNA

1. restrictive enzyme digestion → agarose gel electrophoresis → gel denaturation via alkali soak → transfer DNA to membrane capiliary/electroblot → fix DNA on membrane → hybridize labeled DNA probe → stringent washes remove non-specific probe → detect probe signal → interpret band size to map target loci

Northern Blotting

• isolates RNA

1. extract RNA → quantify and verify RNA quality → denature gel electrophoresis → transfer RNA to nylon membrane → immobilize RNA → hybridize with labeled RNA/DNA probe → high-stringency washes → signal detection

Western Blotting

• isolates protein

1. SDS-PAGE separation → transfer proteins → block membrane to prevent backgroun → incubate with antibody → wash away antibody → incubate with secondary antibody → final wash → detect signal → quantify bands and report protein level

Gene Cloning

• apmplifies a specific piece of DNA via a bacteria cell

• DNA is heated to separate 2 strnade, and quickly cooled to allow single-stranded primers to anneal to complementary, heated again to synthesize DNA strands (creates 2 new DNA molecules)

Cloning Vectors

has an origin of replication, one or more selectable markers and recognition sites (for restriction enzymes)

Plasmid Vectors

restriction enzyme cuts plasmid and foreign DNA, sticky ends join foreign DNA and plasmid and nicks are sealed by DNA ligase (ex. lacZ gene)

Expression Vector

• foreign gene may be inserted into expression vector to ensure transcription and translation

• include sequences that regulate desired gene and operon sequences that allow DNA to be transcribed/translated

Gibson Assembly

• pcr based alternative restriction cloning

• determines amount of DNA amplified as reaction proceeds

1. pcr used create region of homology between dNA fragments and cloning vector

2. enzyme removes nucleotides creating 3’ overhangs which pair (homologous)

3. DNA polymerase fills in nucleotides and ligase seals gaps

Sanger Sequencing

• used to sequence first human genomes

• dideoxy sequencing used to isolate DNA fragment, ddNTP is tagged by dye and sequencing reaction is carried out, products are denatured and dNA fragments are isolated, dye is recognized as peak on computer printout

• reaction requires special substrate for DNA synthesis

Illumina Sequencing

• most widely used sequencing

• similar to dideoxy sequencing

• dNTPs are tagged and also has terminator which prevents incorporation of addition nucleotides when incorporated into DNA chain

1. target DNA is cleaved into fragments

2. fragments create clusters of copies

3. fragments denatured and solution of primers, dNA polymerase and special nucleotides is added

4. primer attaches to DNA template and nucleotide is incorporated into newly synthesized strand

5. solution is washed away

6. tag is recognized with lazor and then chemically removed

Third-Gen Sequencing

• determine sequence of single molecules of DNA/RNA

• allows longer fragments to be sequences (produces longer reads)

• simplifies assembly of fragments into complete genome

Pacific Bio-Sciences

• produces very long read lengths

• has higher error rates, slower sequencing time, and more expensive

Nanopore Sequencing

• DNA is passed through hole in membrane (one at a time) and disrupts an electrical current in membrane (specififc shape of nucleotide is recognized)

• allows for longer reads

• has much higher error rate

DNA Fingerprinting

• use of DNA sequences to identify individual people

• each person’s DNA sequence is unique (allows id)

• uses microsatellites/short tandem repeats (STRs): short DNA sequences repeated in tandem which are found at many loci throughout human genome

• people vary in number of copies of repeats

• STRs are detected with PCR using primers to amplify DNA → length of amplified segment depends on number of repeats

• can be used even if sample if very small

Forward Genetics

traditional approach to study gene functions begins with id of mutant organisms

• used by finding individuals with hereditary derfects (like mice). mutations causing problems can be mapped (genes can be isolated/sequenced)

Reverse Genetics

• begin with genotype/DNA sequence and proceed to phenotype by altering sequence

• genes can be induced by mutations and observed to see effect on phenotype

Transgenic Animals

• adding dNA sequences of interest to genome of organisms that typically lacks sequences and observing effect of introduced sequences on organism’s phenotype

• form of reverse genetics

• in injected eggs, copies of cloned DNA integrate randomly into one of chromosomes through nonhomologous recombination (often use CRISPR-Cas)

• through interbreeding, a strain of mice that carry foreign gene can be created

Knockout Mice

• varient of transgenic approach

• normal gene isn’t mutated but fully disabled

• helpful in determining function of gene → phenotype of knockout mouse gives good indication of missing gene

Knockin Mice

• varient of kockout procedure

• inserts a DNA sequence at known chromosome location

• can make mouse model of human diseases by isolating and inbreeding mice with naturally occuring mutation and using knockin techniques to modify genes

RNAi Silencing

• can use siRNA and miRNA to control gene expression

• turning gene off and observing effect of absence of gene product on the phenotype

Specialized Bacteria

• bacteria can be used to produce ethanol, lead of minerals, and waste treatment

• bacteria is genetically engineered to work efficiently

Agricultural Products

• recombination DNA used to create crop plants and domestic animals with valuable traits

ex. can be modified to be resistant to viruses or pests

• could be used to increase yields in unfavorable growing conditions (meet future need)

Gene Therapy

• direct transfer of genes into human patients to treat disease

• experimental treatment for genetic diseases, cancer, heart disease, aids, etc.

• significant problems still exist

• only targets nonreproductive cells

Structural Genomics

study of complete set of DNA sequecnes and organization of entire genomes

• want to sequence entire genomes to provide info

• eukaryotic genomes are too large to sequence entire chromosome straight thourgh

• can be used to locate genes that affect diseases

• concerns about being used for discrimination/eugenics

Map-base Sequencing

1. partial digestion of DNA results in overlapping fragments which are cloned

2. clones are analyzed for markers to allow large-insert clones to be assembled into continuious stretch of DNA

3. subset of overlapping clones are selected and fractured and cloned

4. small-insert clones are assembled in correct order

Shortgun Sequencing

1. genomic DNA is cut into numerous small overlapping fragments

2. fragment is sequenced

3. overlap in sequence is used to order clones and genomic sequence is assembled by powerful computer programs

SNPs

• arose once from single mutation and spread through population

• initially associated with other SNPs that were present on particular chromosome

• specific set = haplotype

• SNPs in a haplotype are phyisically linked and inherited together

• valuable markers in linkage

• location of disease causing gene can be determined

GWAs

• diseases are caused by complex interactions among multiple genes

• used to find genes of interest

• use SNPs to locate genes that contribute to complex diseases

• genes often explain only a small proportion of genetic influence on trait

• missing DNA sequences (dark matter of genome) remaine undetected

Metagenomics

• genome sequences of groups of organisms inhabiting a common environment are sampled and determined

• extracts DNA from environement, determines sequences and reconstructs community composition and function

Synthetic Biology

• ability to sequence and study whole genomes by creating novel organisms from scratch

• design organisms that can provide useful functions

• creates potential for synthesis of dangerous microbes that can lead to ecological havoc (biological warfare)

Functional Genomics

• identification of RNA molecules transcibed from a genome

• allow gene function to be identified from DNA sequence alone

• homology search = relys on comparisons of DNA and protein sequences from same organisms and different organisms

• can be used to identify functions of unknown genes

Microarrays

• nucleoic acid hybridization uses DNA fragments as probes to find complementary sequences and then ordered in an array

• used to correspond to known genes

• can be converted to a heat map and used to find gene expression

RNA sequencing

• determines presence of RNA molecuels in a cell by sequencing cDNAs copied from cellular RNA molecuels

• provides detailed information about gene expression: types and number of RNA molecuels produced by transcription and presence of alternatively processed RNA

Comparative Genomics

• can allow inferences about how genes function and evolve

• provide evolutionary relationship among organisms and influencual factors