Genetics Exam 3

what phase does DNA replication occur in?

S phase

DNA is considered semi-conservative, what does that mean?

the replicated DNA strands will have one strand directly from the original

where does replication begin in the DNA strand?

the origin of replication (middle of the replication fork, point where two strands first separate)

how many origins of replication do prokaryotes have?

one - genomes are one circular molecule

how many origins of replication do eukaryotes have?

thousands of origins - larger genomes, make replication faster

what proteins are needed for DNA replication in eukaryotes? describe each of their roles

DNA polymerase - synthesizes new DNA

primase - creates RNA primer

single stranded binding protein - holds the two template strands apart

helicase - pulls apart the double stranded template

gyrase - prevents tangles of DNA ahead of helicase/replication fork

ligase - anneals fragments into one DNA molecule

telomerase - prevents the shortening/loss of DNA at the end of the chromosome

(PPSHGLT - pretty please seek help genetics literally traumatizes)

how does telomerase function?

the RNA primer (from primase) is removed from the last okazaki fragment new strand, leaving a gap

telomerase has associated RNA that complements the 3’ overhang, it then attaches to the overhang on the template strand and is used to elongate the template strand

telomerase moves further down and continues to elongate, while DNA polymerase and primase synthesize the complementary strand

what complex is created in G1 phase before replication? what does it do? what other complex does it prepare for?

the origin recognition complex - binds to origins of replication, docking station for

pre-replication complex - group of 20+ proteins that prepare to unwind DNA in S-phase (activated in S phase then disassembles until next G1)

what does DNA replication require?

RNA primers (primase)

which direction does DNA synthesis occur in?

5’—3’ (added onto the 3’ end)

where are the phosphodiester bonds formed? what forms them?

between the phosphate group on C5 and the hydroxyl group on C3

formed by DNA polymerase

what type of enzyme is DNA polymerase considered?

a holoenzyme - made up of multiple protein subunits

how many DNA polymerases are in prokaryotes compared to eukaryotes? give examples

prokaryotes have one - polymerase III

eukaryotes have many (at least 14) - alpha, delta, and epsilon

describe the alpha, delta, and epsilon DNA polymerase functions

alpha - primase, synthesizes RNA primer

delta - lagging strand synthesis

epsilon - leading strand synthesis

RNA primer is ____ to the template

antiparallel

how does the lagging strand differ from the leading strand?

lagging - requires multiple RNA primers (moves in opposite direction as fork), discontinuous synthesis from okazaki fragments

leading - continuous synthesis, occurs in same direction as the replication fork

what is the main goal of PCR?

to amplify a specific region of DNA

what is required for PCR?

template DNA - target for amplification

Taq polymerase - synthesizes DNA

dNTPs - DNA building blocks/base pairs

buffer - creates stable environment for polymerase (pH, salinity)

primers - one forward and one reverse, made of DNA sequences complimentary to the template you want to amplify, initiates synthesis

what are the steps of PCR? where do these steps occur?

in the thermocycler: (repeat 25-30 times)

denaturing - 94-95 degrees, takes double stranded DNA to single stranded DNA

annealing - 45-65 degrees, primers bind to the template

extension - 72 degrees, Taq polymerase synthesizes DNA starting at the 3’ end of the primer

what are the limitations of PCR?

the size of the product - can only amplify a couple thousand nucleotides, contamination sensitive, and you have to know the region of DNA you want to synthesize for the primers

what are the applications of PCR?

genotyping, cloning experiments, identifying contamination in food, identifying pathogens, genetic testing

what are restriction enzymes and how do they function?

enzymes or proteins that cut/digest double stranded DNA at specific recognition sequences

digest = break phosphodiester bonds in DNA backbone (in both strands)

recognition sequences = 4-8 basepairs long

what is the product after restriction enzyme digest?

DNA fragments with either sticky ends or blunt ends

sticky ends = DNA fragments with overhanging DNA

blunt ends = even cut between two strands

how are restriction enzymes used in cloning?

used to facilitate ligation

ligase will repair phosphodiester bonds between fragments, binds sticky ends together

what does cloning produce?

recombinant DNA = DNA from two (or more) different species

INSERT + VECTOR

how does cloning occur?

recombinant DNA is placed in a host cell to replicate it

host cell undergoes DNA replication to make a lot of copies

what is a vector?

DNA molecules that accept foreign DNA fragments to replicate them in a host cell

what are the three types of vectors?

plasmid - (pMiniT) designed for prokaryotic host cells, bacteria (E. coli) contain origin of replication specifically for prokaryotes *6-8,000 basepairs and inserts up to 10 kb

yeast artificial chromosomes (YACs) - host cell is yeast (single-celled eukaryote) *inserts 100-1,000 kb

bacterial artificial chromosomes (BACs) - host cell is bacteria insert *insert 100-300 kb

what are the steps in a cloning experiment?

prepare the insert and vector for ligation - amplification through PCR, restriction enzyme digest to make sticky ends

ligation - recombinant DNA is created using ligase, base pairing between sticky ends bring fragments together, ligase creates phosphodiester bond

transformation - host cells take up the recombinant DNA from ligation, different types of host cells - depends on vector and DNA size

selection - used to determine which host cells have taken up recombinant DNA, carried out with selectable markers, plasmids engineered to contain specific sequences

what are the 2 methods of transformation in cloning?

heat shock - treat competent cells with brief heat shock/exposure to heat, commonly 42 degrees for 30 seconds, plasma membrane becomes permeable and cell takes in DNA

electroporation - short electrical shock (stresses cell), permeable membrane takes in DNA, gentler and more efficient than heat shock but hard to do in large group and need special equipment

what is the process of selection in cloning?

antibiotic selection: place cells on media with ampicillin (cells with plasmid survive and cells without die off)

selects for an insert in the multiple cloning site (MCS - contains several restriction enzyme sequences, where insert is ligated): selects for successful ligation, the presence of the insert will disrupt the toxic mini gene present in the plasmid OR present as white colonies in blue/white selection (Lacz gene)

true or false: transformation in cloning guarantees all host cells took up the recombinant DNA

FALSE - some host cells may not have any, some may have a plasmid without an insert, and some may have the recombinant DNA. we see the results of this through selection (host cells with no plasmid die off) (host cells with plasmid only will either die off or present as white colonies)

how does Sanger sequencing work?

relies on terminator base pair (ddNTPs - no hydroxyl on C3, prevents nucleotide from binding to anything else further, fluorescent tag unique to each basepair)

perform PCR using normal dNTPs and ddNTPs, creates pool of fragments of different sizes and labels

products run through capillary tube to separate them by size (smaller first, software makes chromatogram of order of nucleotides/order of sequence)

how many reads can sanger sequencing produce? how many sequences at a time?

up to 1,000 basepairs

one sequence at a time

cheap - $5

how does sequencing by synthesis work?

PCR based with fluorescently labeled dNTPs

take place in flow cell - bind template strands to flow cell

fluorescently labeled dNTPs are washed over the template one at a time and if the dNTP is complementary, it will fluoresce

order of the fluorescence is the sequence of basepairs (cycle repeated 50-150 times)

computer will compile sequence data for analysis, makes enormous amounts of data

how long of reads can sequencing by synthesis read? how many sequences?

50-150 bp long

can produce hundreds of millions of sequences at a time

3-5k per run

how does nanopore sequencing work?

NOT PCR based, uses synthetic membrane with a tiny hole/nanopore

membrane has set charge on either side

template is fed through the pore and each basepair changes the electrical current in a specific way, the computer records the changes and shows the order of the sequence

very portable (usb drive)

how long of reads can nanopore sequencing read? how many sequences?

very large sequence reads, up to 25 kb

hundreds of millions of reads per run

3-5k per run

what is shotgun sequencing?

method used to sequence whole genomes of organisms

cut DNA with restriction enzymes to sequence the fragments, then use computer software to align overlapping fragments to create a linear sequence of whole genome

what is a contiguous sequence/contig from shotgun sequencing?

series of overlapping DNA fragments

contig = overlapping nucleotides, have the same sequence where they overlap

what is the human genome project?

the first major application of whole genome sequencing on a massive scale

sequenced the entire human genome by 2003 (billions of dollars and carried out with 18 other countries)

what are the two strategies for whole genome sequencing?

clone-by-clone sequencing (from BACs - DNA cut into large fragments, each BAC carries a piece of human DNA, bacterium copies its BAC and now there’s many copies, then map)

shotgun sequencing

what are features of the human genome?

3.1 billion nucleotides that are 99.9% identical between individuals

roughly 22,300 genes

2% of the sequence encodes the gene

40-50% of the gene are repeats

40% of genes have an unknown function

what are bioinformatics?

analysis of data, utilizes statistics, computer software (coding hardware), intersection between biology and computer science/math

use of computer software, hardware, and mathematical approaches to analyze data related to biological questions

ask questions about DNA sequence, gene structure and expression, and protein structure

what is genome annotation?

following whole genome sequencing, need to predict gene location and function/identifying where genes are in a DNA sequence and predicted what they do

scan for start codons (TATA box), read codons in that frame, look for stop codon

use BLAST to confirm gene identity/function

how is BLAST used for genome annotation?

BLAST compares DNA to DNA, DNA to database of known genes, or protein to protein

(not just database of humans, any known gene from a species is compared)

compares individual sequences to a database, can compare two sequences pairwise or compare sequence to whole database

NCBI - organization, US government bioinformatics organization

GenBank - largest sequence database, contains all public sequences, chromosome maps, DNA/RNA/amino acid sequence, expression data, etc.

what is genomics?

the study of all DNA in a cell or organism, study of the whole genome

what is proteomics?

the study of all proteins in a cell

what is transcriptomics?

the study of all RNA in a cell/gene expression

what is metagenomics?

the study of all genomes found in an environment

what are functional genomics? what is genome annotation?

genome annotation - predicting gene location and function, predict the function of all genes in a genome

studying what genes do and how they are expressed/regulated across the entire genome (can be predicted using closely related species/model organisms)

what are orthologs? what are they part of?

functional genomics

evolutionarily related genes in DIFFERENT species (SRGAP2 gene in nonhumans and humans)

what are paralogs? what are they part of?

functional genomics, evolutionarily related genes in the SAME species (SRGAP2 gene - humans have 4 copies)

what is comparative genomics?

compares genomes from different species to ask biological questions

evolution, gene function, expression, etc.

humans share ~50% of our genes with other organisms

what is the first example for comparative genomics?

dog genome - sequenced in 2005, ~2.47 million basepairs, 18,400 genes

60% of inherited diseases in dogs are shared with humans (behavioral conditions, cancer, aneuploidy)

inbreeding has reduced genetic diversity making it easier to study certain genetics (easier to trace)

what is the second example of comparative genomics?

sea urchin genome - sequenced 2006, 814 million basepairs, 23,500 genes, share ~7,000 with humans

large number of genes involved in immunity and “senses” (light detection, odor, hearing, balance)

what is genetic engineering?

alterations of an organism’s genome using recombinant DNA (*cloning!*), uses DNA from other species to alter a genome

what does transgenic/GMO mean?

that organism contains DNA from a different species

what is biotechnology? what are some examples?

the use of living organisms to produce a product

pharmaceuticals, beer, wine, cheese, honey

*doesn’t always involve genetic engineering

what are biopharmaceuticals?

the product of recombinant proteins used to treat a human disease/disorder, very common for genetic engineering (NOT the host cell, the product of the host cell → insulin)

human clotting factor → expressed in sheep (transgenic)

extracted from animals/plants prior to GE

what is biopharming?

the production of biopharmaceuticals using whole genetically engineered plants or animals

how the drug is produced → use plants/animals as factories to produce pharmaceutical proteins

(insert gene is delivered into plant/animal, organism produces a protein, protein is extracted and purified, then the drug is administered normally)

what is an example of biopharmaceuticals?

insulin - prior to GE, extracted from pigs

now produce a human protein through recombinant insulin from cloning

(prepare vector and insert, ligate coding sequences/genes for human insulin protein A and B into a plasmid, transformed plasmids into E. coli, purify and mix protein subunits A and B together to make active insulin

first GE product approved use in humans, FDA 1982

how do biopharmaceuticals relate to vaccines?

some vaccines are produced with GE (not traditional vaccines)

they expose the immune system to pathogens to produce antibodies

what are the two types of vaccines that do not use GMOs?

inactivated - contain whole organisms that are dead (virus or bacteria), like flu/rabies

attenuated - contain whole living organisms/pathogens that have been altered to prevent infection, like chicken pox/cholera/typhus

what are the other two types of vaccines that do use GMOs?

subunit vaccines - consist of only surface protein from a virus or bacteria, use cloning to express the gene for this protein in a host cell, protein is purified and used as an injectable/edible vaccine, like hepatitis B

mRNA vaccines - vaccine consists of messenger RNA that will be translated into protein in the individual, a gene sequence for a coat protein - protein from a pathogen, into a plasmid and express RNA in a host cell, RNA is purified and used as injectable vaccine, only COVID vaccine is approved (mRNA is severely unstable and degrades, must be kept at low temperature and bound otherwise lipid nanopore for stabilization)

what are edible vaccines? how do they relate to subunit vaccines?

they use cloning to produce a protein from the pathogen in plants (there’s a GMO plant that expresses the protein)

in production, not FDA approved

can be more accessible in less developed countries but how will it be regulated? how will dosing work?

describe genetically engineered animals in regard to agriculture, recreation, food, and the environment

most GE animals are created for research purposes and pharmaceutical production (biopharming)

ag: used to prevent disease, animals are transgenic to express a protein to prevent bacterial infection (mastitis/bacterial infection in cow udders)

env.: enviro pig - pig farming creates lots of phosphorus, transgenic animals created to secrete enzyme in saliva that allows them to digest food differently without phosphorus

rec.: glofish - transgenic zebrafish that express fluorescent proteins, just for fun

food: AquaAdvantage salmon - transgenic fish that express growth hormone from Chinook salmon that makes them mature faster (18 months to 3 years)

how were plant varieties developed before GE?

selective breeding - when humans select for a desirable trait over many generations until it’s consistent in that species (domestication of corn from teosinte)

requires genetic diversity or trait already being present so it can be selected, takes a lot of time (15-20 years)

why was GE in plants used?

it introduces traits very quickly, only takes 1-2 years

first approved plant was FlavrSavr Tomato in 1994

common in US are cotton, soybean, and corn

what are the three common traits found in GE plants?

herbicide tolerance - (round up) resistant to herbicide glyphosate, farmers can apply glyphosate to field and these plants will survive, prevents weed growth, 70% of GMOs have this, can create resistant weeds

insect resistance - plants express protein from bacteria (Bacillus thurengenesis/Bt), protein is toxic to specific insects (moths, bees, mosquitos, butterflies, corn ear worm, cotton bull weevil), insect feeds on GMO plant and protein crystalizes in its gut → death, used in organic farming (applied as dust), not toxic to humans, reduce crop loss to insect damage