molecular biology Flashcards
dna
highly discriminating typing systems
very sensitive ( touch dna, degraded samples, greater stability compared to ecological markers)
highly specific markers which can be targeted
amendable to automation
permits data-basing
conventional serology
relatively poor discrimination
useful for exclusions
low power of discrimination Pd
required visible blood and relatively large sample
power of discrimination
definition: probability of discriminating two distinct samples selected at random from the population of interest
power of discrimination increases as you go down
warm vs cold blooded
2 vs 4 legs
skin vs hair on body
male vs female
what color hair
what color eye
prokaryotic cells
lack a membrane bound nucleus and other organells
bacteria are prokaryotic unicellular microorganisms
eukaryotic cells
eu=true
interior of cell is organized into many specialized compartments or organelles each surrounded by separate membrane
plants and animals are eukaryotic multicellular organisms
dna
deoxyribonucleic acid
can be found in these cell types
blood, muscle, bone marrow, tooth pulp, hair roots, saliva, semen, tissue
items
chewing gums, envelopes, stains, washed stains, doorknobs, toothbrushes, sanitary pads
diploid
somatic cells
bodily
2 sets of each chromosome 46 total, 23 pairs
dna=6.4 billion base pairs
6.6 pg of DNA/cell
mitosis
all nucleated cells in the body except egg and sperm
haploid
gametic cells
sex
1 set of chromosomes
23 unpaired total
dna=3.2 billion base pairs
1/2 of diploid
meiosis
only egg (ova) and sperm
mitosis
prophase
chromosome duplication (4n)
duplicated chromosome (2 sister chromatids)
metaphase
duplicated chromosomes align at metaphase plate
anaphase telophase
sister chromatids separate and move to the opposite poles
2 diploid daughter cells (2n each)
meiosis
meiosis I
prophase I
chromosome duplication (4n)
tetrad (two pairs of sister chromatids)
metaphase I
tetrads align at the metaphase plate
anaphase I telophase I
homologous chromosomes separate and move to the opposite poles
two haploid daughter cells
half the number of chromosomes, but same number of sister chromatids as parent cell
meiosis II
anaphase II
sister chromatids separate
four haploid daughter cells
half number of chromosomes
half number of chromatids as parent
sperm
sperm head is difficult to break open because very dense with disulfide bonds
hard nut to crack
differential extraction
non sperm dna first
then sperm dna
exploits this
mitochondria are inherited from mother
father mitochondrion breaks off as sperm head enters egg
how can you get a full dna profile if sperm only contains 1/2 dna
all sperm cells have diff combinations of alleles
in totality it will give you a full profile
diploid cells
two types of dna
mitochondrial and nucleic
dna in blood
platelets and rbcs don’t have nuclei therefore no ndna
white blood cells have ndna
mitochondrial circular dna genome
each mitochondria has 2-10 copies of mtdna
only maternal mtdna is inherited
hypervariable regions I and II are used for forensic comparisons
number of mtdna»_space;> nDNA in terms of copy number
cell with many mitochondria only 1 nucleus
ndna is much larger than mtdna, degradation
high probability of degraded dada in a degraded sample but some mtdna probably undegraded
is your dna the same in all nucleated diploid cells
no
polymorphisms, mutations, telomere degradation
however, the forensic dna primers target regions of repetitive dna and we only measure the length of those segments so these are the same
unless in very rare situations
regions that don’t change therefore are the same throughout individuals entire life
what is the forensic advantage for having the forensically speaking same dna in every cell
we can obtain the same profile regardless of the tissue source and the age of the sample
blood or semen as evidence can be compared to a buccal swab
or evident from an old crime scene can be compared to a buccal swab collected at a much later time
if your dna is the same in every cell, why don’t all the cells look and act the same
not all cells express turn on the same genes
many genes are tissue specific and there are many factors outside of genes that regulate when a gene is turned on or off
independent assortment in meiosis leads to genetic variation
first glimpse of genetic variation independent assortment, random
with independent assortment at meiosis I
a single individual can produce 2^23 or 8.4 million different combination of chromosomes in a haploid cell
with fertilization of the egg by the sperm, there are over 70 trillion (2^23x2^23) possible combinations of chromosomes
crossing over increases genetic variation
prophase I
homologous chromosome pair
4 pairs of sister chromatids
as the chromosomes move closer together
synapsis occurs
chromatids break and genetic information is exchanged
also called genetic recombination
linkage equilibrium =. inherited independently
alleles are considered in disequilibrium when they are connected (always inherited together) , but crossing over can disconnect alleles not same chromosome
chromosomal rearrangements
meiotic nondisjunctions
when one or more chromosomes fail to separate during either meiotic division
trisomy 21 Is down syndrome
sex chromosome aberrations
XO - female, Turner syndrome
XXY - male, Klinefelter syndrome
XXX - female, trisomy X
duplications deletions and inversions
duplication is a copy number variant can be lethal when by essential carefully regulated genes, or have no affect at all
2 allele pattern in STRs
deletions loss of genetic material
unless confined to small or inessential area, usually lethal
inversions - breakage of a chromosomal region followed by rejoining in the reverse orientation
translocation - transfer of a region on one chromosome to another
structure of dna
bases sugar backbone base pairing double helix
5 atoms C, N, H, O, P
structure of dna supports its function
genetic expression
to code for proteins needed for survival
replication (in mitosis)
to propagate the hereditary material during development of an individual
recombination (meiosis) to shuffle hereditary material between successive generations
these are exploted by forensic dna analysis
nucleotides and polynucleotides
dna is antiparallel
ndna is a linear polynucleotide
polynucleotide = individual nucleotides linked by phosphodiester bonds
each nucleotide contains 3 componenets
a deoxyribose, sugar
a nitrogenous base
a phosphare grou
4 bases: char gaff’s rules
adenine, cytosine, guanine, thymine
CG stronger pairing than AT 3 H bonds
pyrimidines (1) and purines (2)
dna the double helix
described by Watson and crich received the 1962 noble proze
3 forms, b-dna most commonly found in nature
complimentary: TA and CG
antiparallel, 3’-5’, 5’-3’
stabilized by chemical reactions
base pairs - hydrogen bonds provide weak electrostatic attraction between electronegative atoms
base stacking
involves hydrophobic interactions between adjacent base pairs, provides stability to double helix
van der Waals forces - aromatic ring stacking forces vetween base oairs
hydrophobic effects
non polar, uncharged bases are present in the interior of structure while negatively charged phosphates are on the outside
important because in aqueous environment
organization of dna into chromosomes
most basic complex of the chromosome is the dsdna
dsdna is wound around proteins called histones
small +. charged proteins
because phosphate backbone is negative
nucleosomes are made up of dsdna complexed with hsitones
further compacted into chromatin fiber
each chromosome contains a large number of looped domains of chromatin fibers attached to a protein scaffold
denaturation
occurs when hydrogen bonds between base pairs are disrupted and strands separated
melting curve
obtained by measuring dna denaturation by slowly heating a solution of dna
melting temp tm the temp at which 50% of dna strands is denatured
salt conc can affect tm
nucleotide content can affect tm lots of CG increases TM because there’s more H bonds
length of molecule can affect tm
ph can affect tm
renaturation
also called reannealing
under certain conditions single strands of dsdna can reform
2 requirements much be met
salt conc high enough to neutralize the negative charges of the phosphate groups that would otherwise repel each other
temp must be high enough to sufficiently disrupt h bonds that randomly formbetweenm bases of the same strand, pinloop
but not too high that they can’t form between complimentary strands
dna replication
dna polymerase
an enzyme that synthesizes long chains of nucleic acids always moves in 5’-3’ direction
synthesis is continuous on the leading strand and discontinuous on the lagging strand, creating Okazaki fragments
dna replication requires a primer
a short single stranded DNA sequence that is complimentary to a specific target sequence of dna
primers indicate the starting point for the synthesis of the new strand by the polymerase enzyme
dna replication
teamwork makes the dream work
in vivo
gyrase, topoisomerase
relieves positive supercoils ahead of fork, physics
helicase
unwinds the dsdna helix
dna primase - a type of RNA polymerase which catalyzes the synthesis of a short ran primer complimentary to ssdna
dna polymerase II synthesizes dna using ran primers
dna polymerase I removes the primer on the lagging strand and replaces it with dna
dna ligase seals gaps between Okazaki fragments
dna synthesis occurs at multiple locations at a time in vivo
bubbles and forks meet each other
coalesce
all polymerases rent created the same
dna polymerase characteristics
initiation/regulation how and where polymerization is activated and how its cntrolled
polymerization rate - number of nucleotides polymerized per second
error rate - # of incorrect nucleotides
processivity number of nucleotides added per binding event
proofreading functions - does it have an error-correcting function
primer specificity
ABI Amplitaq Gold was gold standard
has a hot start modification added to allow room temp set up
optimal operating temp is 72
dna replication in vitro: pcr
utliizes synthetic primers to target specific areas of the genome
uses the polymerase chain reaction
makes millions of copies of target sequences from one strand of dna
pcr
temps not specific, depends on dna
3 basic steps
denaturations (96) heat is added to separate or denature dna strands, providing ss dna templates
annealing (55-65) reaction is cooled to allow primers to bind to their complimentary sequences
depends on the tm of primers
extension 72 temp is raised again so that taq polymerase extends the primers, synthesizing new dna strands (specific temp is based on the optimal operating temp of polymerase being used)
repeats the steps 25-35 times, 2- 4 hours
not just the original dna is used as a template
each cycle uses the new dna strands in addition
copies of copies
pcr reaction components
reaction buffer (salt)
reduces Tm of DNA because negative charge of the phosphate backbone
MgCl2 improves primer binding
primers - target areas of DNA and primes the synthesis of DNA
dNTP’s
Polymerase
template DNA
Let’s talk chromosome
each chromosome has a short arm (p) and a long arm (q) separated by a centromere
centromeres are the dense DNA sequences found near the points of attachment of mitotic or meiotic spindle fiber
telomeres are the ends of the chromosomes, help stabilize chromosome and play a role in replication
cytogenetic mapping - chemical staining of metaphase chromosomes resulting in an alternate banding pattern, can be seen under a microscope, used to identify locations on a chromosome
ex: AmelY (amelogenin, Y-linked) is Yp11.2 which indicates its location on chromosome Y, short arm, band 11, sub band 2
gametes are haploid (22 autosomes + 1 sex chromosome)
in ova always X
in sperm can be X or Y
formed by germ cells
fertilization results in a zygote
diploid
most somatic cells are diploid
some hav eno nucleus hair epithelial skin nails
result = 46 chromosomes
homologous chromosomes the two chromosomes of a pair in a diploid cell, one inherited from sperm, the other egg
karyogram shows chromosomes
the structure of dna supports its function
genetic expression
to code for proteins needed for survival
replication (mitosis)
to propagate the hereditary material during development of an individual
recombination (meiosis)
to shuffle hereditary material between successive generations
these are exploited by forensic dna analysis
chromosome: locus and allele
locus (plural loci)
the chromosomal location of a dna marker in a non-coding region
gene: the segment of dna which codes for a functional ran or a protein product
allele
the alternate form of a gene or genetic locus
homozygous: identical alleles at a locus on homologs chromosomes
heterozygous different alleles at a locus on homologous chromosomes
genotype (spelling)
the characterization of the allies present at a genetic locus
phenotype (what it is saying)
the physical manifestation of a genotype: visible trait
human nuclear genome
3.2 billion base pairs (haploid)
genes and related sequences + intergenic noncoding sequences
human genome project initiated in 1990 and completed in April 2003
genes and related sequences
25 % human genome are genes or related sequences
1.5% is coding DNA ~25000-30000 genes
23.5% non-coding regions (promoter, introns, pseudogenes, etc.)
encode the information for the synthesis of proteins
most human genes are discontinuous
exons, introns
during gene expression the primary mRNA contains both introns and axons throughout the process of splicing the introns are removed and the axons are joined together
messenger RNA
can be used for protein synthesis via translation process
central dogma theory
DNA
transcription and mRNA processing
mRNA
translation
protein
post-translational modifications
active protein
intergenic noncoding sequences
75% of human genome
located between genes
20% is single copy sequences with unknown function
55% is repetitive DNA
2 categories
Intersprersed repeats
randomly located throughout the genome
SINEs - short interspersed elements ~300kb
LINEs - long-interspersed elements ~ 6-8kb
LTR elements - long terminal repeats, telomeric regions
DNA transposons (floating)
or tandem repeats
smaller, back to back
satellite: centromeric, long chunks
mini satellite: forensic VNTRs (variable # tandem repeats) and RFLP
micro satellite: Forensic STRs
forensics utilizes mini satellite and micro satellite
tandem repats
mini satellites - variable nucleotide tandem repeats (VNTRs_
banding, first DNA typing
located in subtelomeric region end of chromosome
core repeat size = 10-100 bp
overall size = 500 bp-30kb
repeated in tandem, over and over
obey hardy Weinberg equilibrium
example D1S80 - 16bp repeat
OJ Simpson one of 1st DNA cases
size not ideal for forensics, very long
not good with degraded DNA
need a large amount of sample
50-1000 ng DNA needed
micro satellites - short tandem repeats (STRs)
~3% of genome, dispersed throughout )genie and extragenic)
core repeat size - 2bp-6bp (1/2 size of smallest VNTRs
overall size = 50bp - 350bp
obey hardy Weinberg equilibrium
example: original codes 13 loci
smaller size, large # in genome, polymorphic (diff alleles, diff versions at different loci)
multiplexing capabilities, PCR with diff primers, all in 1 chem reaction
HWE made ideal for forensics
.5-2ng DNA needed
extragenic dna
forensically interesting regions
types of markers: length and sequence polymorphisms
polymorphism the existence of two or more alleles at significant frequencies in the population
repeat length polymorphisms: STR/VNTR
sequence variation: single nucleotide polymorphism (SNPs)
diff version, same loci
single nucleotide polymorphisms
SNPs
substitution of a single nucleotide, point mutation
occurs ~ every 1000 bases
markers for ethnicity, phenotype, lineage, and identity
three types of str loci
length, repeats
simple, compound, or complex
1st generation forensic dna technologies
RFLP: restriction fragment length polymorphisms
1st described by Alec Jeffreys PhD in 1985 and used in forensic casework in 1986
Pitchfor murders in Leicestershire England
Colin Pitchfork was identified as a primary suspect when he asked a friend for a blood sample
uses restriction endonuclease - cuts DNA at specific target sequence
size polymorphism
VNTR, minisatellites
per based methods
DQ-alpha polymarker
sequence polymorphism
reverse dot blot
D1S80
VNTR size polymorphism
PolyMarker
6 plex PCR for SNPs
Singleplex STRs with silver staining
RFLP assays
banding
no statistics
good for exclusions
polymarker assay
papers with dots that turn a certain color for positive
PCR on each dot location with specific primers
not multiplex
DNA typing today: STRs
of repeats indicates allele
an accordion like DNA sequence that occurs between genes
number of consecutive repeat units can vary between people
FBI selected 13 core STR loci that must be run in all DNA tests in order to provide a common currency with DNA profiles
dna typing today: STR-PCR why its so sensitive
28 cycles
2^28 from one strand of ds DNA
over 268 mil copies from one dsDNA
<1ng target DNA
we don’t need that much
quant system goes down to 50pg, about 9 haploid cells
electropherogram
multiplex PCR
fast and efficient, robotic friendly
very sensitive <1ng DNA
high power of discrimination PD>10^21
DNA profile is 23 autosomal STR loci
Promega PowerPlex Fusion 6c
ABI GlobalFIlerTM
polymorphic, diff alleles per loci
loci - locations on chromosomes
so you can see differences in genetic profiles, but how do you give it weight
what’s the frequency of this profile in a population
mathematically speaking
Mendel’s laws of inheritance
1st law of segregation
diploid organism passes a randomly selected allele for a trait to its offspring
such that the offspring receives one allele from each parent
half the parental gametes carry one allele
and the other half carry the other allele
2nd law of independent assortment
the alleles of two or more difference genes get sorted into gametes independently of one another
the alleles;e a gamete receives for one gene does not influence the allele received for another gene
hardy-weinberg equilibrium
p^2 + 2pq + q^2 = 1
1 in population
p^2 is dominant heterozygous frequence
q^2 is recessive homozygous frequency
2pq is heterozygous frequency
population assumptions for equation to be true
random mating
no natural selection
large population, no isolation, lots of possible allele combinations
no mutation/migration no new alleles being introduced or leaving
allele frequencies remain constant generation to generation
determining freq of alleles
Mendel’s laws of segregation nd independent assortment are the basis for linkage equilibrium and hardy-weonberg equilibrium
HWE deals with mendelian genetics in the context of populations of diploid, sexually reproducing individuals
p^2 + 2pq+ q^2 = 1 are the expected genotype frequencies in a random-mating population for two alleles
these are tested when creating dna population databases
linkage equilibrium alleles
genes in random association
inherited independently of each other
Punnett square
each parent could five either an A or a allele to the next generation in the gamete
there is an equal chance for giving either
1st law of segregation
p+q=1 allele frequency
(p+q)^2 = p^2 + 2pq + q^2
genotype frequencies
HWE is a simple way to relate allele frequencies to genotype frequencies
how statistical calculations are made
generate data with set of samples from desired population groups
generally only 100-150 samples are needed to obtain reliable allele frequency estimates
determine allele frequencies at each locus
count number of each allele seen
there is a mathematical eq to deal with new alleles
migration of new allele
mutation
allele frequency info is then used to estimate a genotype freq at a locus and the rarity of a particular DNA profile
homozygous (p2) and heterozygous (2pq)
use product rule (multiple locus freq estimates)
apply to all loci
forensic dna wouldn’t be complete without codis
13 codes core str loci
D13S317
chromosome 13
317th location on chromosome that they looked at to determine if it was k for forensics
not how chromosomes should be labeled
codes core 20 loci effective jan 2017
codes core 13 t minimum is required for NDIS upload
any 13 core 20 loci
