Molecular Genetics Exam 1

genome

nucleic acid sequence that encodes info for production of an organism, genetic material transferred to new gens

deciphered using DNA sequencing and assembly, manipulated using CRISPR, mutational and transgenic methods

genes

functional units arranged linearly on chromosomes

stable, changes arise from errors of replication and mutations (driving evolution)

genotype vs phenotype

genotype is complete set of genes (alleles) and phenotype is observable characteristics (trait)

human genome

3.2 billions bases

largest genome sequenced was Tmesipteris oblanceolata (fork fern) and smallest Tremblaya princeps (a bacterium)

image stitching

combine 2 or more overlapping images to make 1 larger image

contig (contiguous sequence)

DNA unit created from series of overlapping sequence that create a map that reconstructs original DNA sequence of a chromosome or regions of DNA, multiple restriction enzymes needed

fragment overlaps need enough basepairs to create unique overlap to form full sequence

4 N-bases and 15-16 bases overlap to ensure single occurrence in human genome 4^15 = enough for human genome

assembling genomes from sequencing genomic library

through cloning bacteria or yeast - recombinant DNA made from fragments and plasmids

through cell-free cloning - PCR

strategies - sequencing fragment ends, entire clones (better if you know where clones come from), entire libraries

assembling individual sequence into genome sequence

clone contig method - older, step wise approach to sequencing clones that have been placed on genomic map using markers

shotgun sequencing - recent, uses computer to find overlaps in large amounts of randomly generated sequence to produce multiple contigs that are assembled onto genomic map

cloned contig problems

cloned contig might not produce whole genome in one contig causing incorrect overlap

tandemly repeated sequences - small sequence repeated may times in one location

genome wide repeated sequences - appear at different locations in genome

genomic libraries

ordered collections of DNA fragments maintained as clones, representative (no missing genome), larger fragments better, cloned DNA easily purifiable for sequencing, can be stored and duplicated

vector insert size

bigger size overcomes problem of repeated DNA, inverse relationship with number of clones

smallest → largest vector - lambda, cosmid and fosmid, P1, BAC and PAC, YAC, Mega-YAC

lambda vector

4.6x genome means library should contain 4.6x genome length to be completely representative

N (number of clones required) = ln(1-P)/ln(1-a/b)

lambda genome has optional DNA that can be replaced with any DNA fragment into impairing lytic growth

78-108% can be packed into capsid, minus not optional DNA to find kbp of inserts - upper limit to fit everything and lower limit so still viable

plasmid vectors

replicate as plasmids, include cosmid, fosmid, P1, BAC, and PAC, each colony contains multiple copies of just one recombinant DNA

origin of replication allows plasmid to replicate independently of host (specific to plasmid), selectable marker provides way to identify and maintain cells taken up by plasmid, MCS has restriction enzyme recognition sites, universal sequencing primer site is DNA flanking MCS

lambda as vector in lytic infection

virulent phage, lambda bacteriophage attaches to E.coli and injects its DNA from head into bacteria, kills host cell, lambda DNA directs synthesis of new phages, cell lysis to release these new phases

cro protein dominates over cI

plaque assay

add drop of lambda in test tube with host cells in warm liquid agar, infected host cells placed onto petri dish with growth medium solidified with agar - measures density of bacteriophage

clear zone of host cells (dots) killed by virus in infection center, hazy area (background) where uninfected hosts grow

growth curve

inoculation - inoculum of virus binds to cells

eclipse - small decrease as virions penetrate cells because it take time for host to produce new phages

burst - sudden increase has host cells release many viral particles

burst size - number of virions released per bacterium, levels off

lambda as vector in lysogenic infection

after lambda DNA injected into cell it is integrated into bacterial chromosome, doesn’t always kill host, return to lytic cycle

wt lambda mixed with cI mutant - cloudy plaque with lysogenes growing (cI mutant cannot form lysogenes)

cI protein dominates over cro

restriction site (R site)

needed for lysogenic growth, when insertion made between two R sites lysogenic cycle abolished, new DNA replaces stuffer fragment

lambda replication

lambda genome circularizes during infection (plasmid replication), after rolling circle replication with cos site as cute site for newly made genome, these linear DNA assembled into phage

replacement vector as cloning vector

cos site is cohesive end site, reversible sticky end that allows lambda phage to switch between linear form (in phage head) to circular form (host cell)

restrict and ligate with DNA to be cloned between lift and right arm, can be packaged into infectious lambda phage

cos L inserted DNA R cos

plasmid problems

large plasmids can’t be efficiently transformed - efficient large plasmids include lambda, cosmid, and P1

high copy number causes repetitive DNA unstable in bacteria - F plasmid has low copy number origin of replication

cosmid

high copy number origin, cos sites

fosmid

F- low copy origin, cos sites

P1

phage 1 based clone, PAC is P1 PAC site, P1 low copy origin

BAC

bacterial artificial chromosome, F-low copy origin, cos site

YAC

linear with centromere (ensures segregation during cell division) middle and telomere (protects ends from degradation) ends that allow construction of cloning vectors as they were characterized

MCS cloning site provides location for DNA fragments and Ori allows YAC to replicate in E coli

stuffer fragment replaced with BamH1 in plasmid, cloning done by restricting with BamH1 and SnaB1 to insert DNA between left and right arms

DNA polymerase

needs single stranded DNA template, dNTPs, primer with 3’ OH annealed to template, where primer is determines which part of DNA is copied, where DNA synthesis occurs 

DNA polymerase I

E. coli enzyme, 5’→3’ DNA synthesis, 3’→5’ exonuclease activity (proofreading if mismatch of basepair), 5’→3’ exonuclease activity (gap repair to clean up fragments)

can be converted to Klenow by proteolytic cleavage using a protease

Klenow polymerase

modified E.coli DNAPI, unable to cleave DNA ahead of it because no 5’→3’ exonuclease (no gap repair), which is good because it causes primer degradation causing less labeled DNA product and less PCR product

Sequenase

T7 DNAP with high processivity, longer and faster continuous DNA synthesis runs because lacks 3’→5’ exonuclease, synthesizes DNA without pause

Taq polymerase

thermally stable at high temps, lacks 5’→3’ exonuclease, good for PCR and DNA sequencing to separate strands

first PCR used Klenow and had to be added after each heating cycle

reverse transcriptase

retroviral DNA polymerase that uses RNA as template to make DNA, used to make cDNA from RNA

PCR

DNA region to be amplified is denatured to separate strands, cooled then primers with 3’OH added, heated a little to allow for DNA synthesis, short product accumulates exponentially

agarose gel electrophoresis done to visualize bands under UV light using size markers

PCR primers

PCR requires 2 primers that go 5’→3’, each primer binds to one of the DNA strands, improper primer selection will not produce PCR product (primers going away from each other), overlapping primers make primer dimer making new band on gel

3’ end mismatch causes no PCR product because DNA polymerase can’t extend, mismatch in middle of primer tolerated and useful for introducing mutations

large insertion and deletions tolerated in primer and useful

linkage analysis

mapping heritable frequency of one phenotypic change relative to a different one, analyze structure of genomes

linked genes in same linkage group, independent transmission of both depends on how frequently cross over occurs between them during meiosis

unlinked genes in different linkage groups, no crossing over occurs, either on different chromosomes or far apart on same one

linkage map

cM (centimorgan)used to measure frequency of genetic recombination, 1cM=1% chance that 2 markers on chromosome will become separated from one another

uses of genomic markers

arranging DNA clones into original context in genome preparation for sequencing, sequence of gene associated with a mapped gene is useful

identification of linked gene like for human disease genes for genetic testing or valuable trait genes for assisted breeding

mutation

change in nucleotide sequence, differences in DNA sequence called polymorphism

SNPs every 10kbp in eukaryotes, makes possible highly detailed maps

RFLP

SNP at restriction site causing two fragments to be one on the other allele

southern blotting used to detect specific DNA sequence, transfer DNA from gel to nylon membrane, hybridization analysis using DNA probe and autoradiograph

PCR made identifying RFLPs easier with primers around polymorphic site, followed by restriction of PCR product

oligonucleotide hybridization

very specific, one mismatch in middle makes hybrid unstable and oligonucleotide doesn’t bind 

terminal mismatch at end still allows it to bind

dye quenching to detect hybridization with quenching compound and fluorescent label at different ends of probe attached to target DNA with SNP, DNA microarray and chips incubated with labeled probe - no ligation in assay if there is mismatch with SNP

DNA ligase

in vivo synthesizes missing phosphodiester bond with OH and PO3-

ARMS test

amplification refractory mutant analysis system, design 2 primers - one at SNP site and another further down on other strand, no PCR product if there is mismatch

tetra-primer ARMS uses 2 outer and 2 inner primers on different alleles, PCR and gel run to make homozygotes and heterozygote - a 1 tube reaction, simultaneous detection of both alleles, high specificity

SSLP

differing repeats in alleles, primers flank genes and more repeats means more PCR product, different density than other DNA

minisatellite (VNTR) are 10-60 units and microsatellite (STR) are 2-5 units repeated 5-50 times - satellite DNA used in genome mapping, forensic and paternity genotyping, agricultural genotyping for patent protection

STS

sequence tagged sites, any DNA sequence ca be used as map markers

RGS are random genomic sequences from sequenced ends of genomic clones or from sequence databases

EST is expressed sequence tags from sequenced ends of cDNAs, also map position of expressed genes

clone library for STS

clone library used as mapping reagent using overlapping clones in microtitler plate

mRNA→add oligo(dT) primer→ first strand synthesis by reverse transcriptase→ ribonuclease H degrades most of RNA leaving fragments attached → second strand synthesis by DNAP1→ completion of second strand synthesis (cDNA cloned into vector)

fidelity vs processivity

fidelity is accuracy of DNA replication (correct nucleotide put in) and processivity is ability of DNAP to add many nucleotides to growing DNA strand

dideoxy chain termination sequencing (Sanger)

uses single stranded identical DNA templates with primers with 3’OH, dideoxynucleotides where OH replaced by H for chain termination, DNA polymerase, all dNTPs - strand synthesis terminates when ddNTP added

low cost and simple data analysis but sensitivity, scalability, and sample input requirement drawbacks

ddNTPs each labeled with different fluorescent are sequenced and detected in imaging system, computer creates graphs with peaks for each bp

early Sanger method

used Klenow (no 5’→3’ exo) than later Sequenase (no 5’→3’ or 3’→5’ exo) so no primer loss and high processivity, ddNTPs radioactively labeled for sensitivity since little DNA made (single primer extension reaction), 4 reactions carried out separately each with different ddNTP but same radio-label

single stranded template problems

secondary structures like stem loops block synthesis, nucleotides read going up but sometimes compression (two bands in one) - resolved by DNA insert that is transfected into E coli that releases phages with protein coat around DNA core (recombinant M13 phage)

current Sanger method

heat stable DNA polymerase, thermal cycler, fluorescently labeled ddNTP, template DNA PCR with one primer than add ddNTP, chain terminated strands increase as more cycles carried out (not PCR)

chain termination method of entire clone

universal primer can be used on every clone in library, internal primer is specific and creates sequence by ends of previous primer overlapping (primer walking)

DNA polymerase extend a DNA chain with ddNTP at 3’ end because ddNTP lacks 3’OH group

not amendable to next Gen sequencing

Human genome project

mapping and sequencing with dideoxy method for accuracy, develop technology to sequence genome and discover gene function

pyrosequencing

pyrophosphate released during DNA synthesis and detected by coupling, if template has C then only dGTP will have chemiluminescence while others degraded and washed off

flowgram used for base calling on amount of light admitted, peak twice as high = 2 same bp in a row

good for short sequences, can produce high amount of sequence using chip

pyrophosphate (PPi)

produced when dNTP into growing strand detected by sulfurylase (PPi + AMP → ATP), Luciferase gives off light in presence of ATP, apyrase added to remove excess dNTPs after each round

Next gen sequencing

library for template DNA fragments → clonal amplification through emulsion and bridge PCR → cyclic array sequencing through pyrosequencing, sequencing by ligation or synthesis

Next gen sequencing using bridge PCR

oligonucleotide adapter (short dsDNA) joined to fragmented DNA by DNA ligase on both sides → melt adapter-ligated fragment so its single stranded and anneal to bead with complimentary primer attached (single DNA to single bead) → perform primer extension DNA synthesis → melt to leave ssDNA fragment covalently on bead → anneal to produce bridge that prime another primer extension → leaves two copies of DNA fragment → repeat annealing and primer extension to have 4 copies of DNA fragment → perform pyrosequencing at same tie

multiplex sequencing

designing different adapters so many DNA samples can be sequenced at same time

ion torrent sequencing

semiconductor chip sequences DNA

sample of DNA cut into fragments → each fragment attached to own bead and replicated until it covers whole bead → each bead is deposited into well on chip → flooded with one type of DNA nucleotide and when a nucleotide incorporated a H+ ion released to change pH changing voltage that is measured to indicate nucleotide → repeated every 15 sec with new nucleotide

if same base pair twice, its compliment will release more H+ which changes voltage more to indicate two bps

good cost and speed by short length reads and accuracy drawbacks

nanopore sequencing

double stranded DNA unwound by motor protein and one strand goes through reusable nanopore underneath into membrane → ions now inside membrane flow out to create ionic current where DNA sequenced as each base gives characteristic reduction in current

done in wells where each well reading different DNA sequence

minION and PromethION, good for long reads, direct sequencing of RNA and detecting RNA modifications, but costly, error rate, and large amount of starting material

clone contig sequencing

arranging library of clones in the context they exist in intact genome before clones are sequenced, macrolevel organization finds clones that contain known makers then work outward from starting point using chromosome walking (finding overlaps)

takes time to organize clones but methodical to make sure as little sequencing done

shotgun sequencing

extract DNA and sonicate (shear) into different size fragments → run on gel with H.flu DNA lane and DNA markers lane → purify DNA from gel (DNA fragments 1.6-2 kb) → prepare clone library and obtain end sequences of DNA inserts → construct sequence contigs

H.flu

genome was first to be sequenced with shotgun sequencing (1,830,000 b genome)

clone library was 20x genome size and 24k successful sequences with 16% failure, 140 separate nonoverlapping contigs, clone library had 99 clones with ends in different contigs, 42 gaps

closing physical gap

contigs probed in second clone library with oligonucleotides at ends, PCR done with pairs of oligonucleotides - primers that hybridize to same clone are part of adjacent contigs

oligonucleotides from contig ends flanking gaps were use to probe clones produced in lambda vector (closed 23 gaps)

PCR methodology for closing gaps

run PCR with 1 insert and oligonucleotides with all other clones, band shows up with overlapping one

combinatorial approach

reduces number of PCR reactions needed

combine all samples in rows and perform PCR, combine all columns and perform PCR, mix corresponding well from each tray and run PCR