Genetics Final

Human Genome Project

an accurate sequence of the human genome

general steps behind genome sequencing

1. fragment the genome

2. clone the DNA fragments

3. sequence the DNA fragments

4. reconstruct the genome sequence from the fragments

restriction enzymes fragment the

genome at specific sites

each restriction enzyme recognizes a

specific sequence of bases anywhere within. the genome

how long are recognition sites for restriction enzymes usually and are often?

4-8 bp and are often palindromic (sequences of each strand identical when read 5’-3’)

restriction enzyme cuts

the sugar-phosphate backbones of both strands, generating restriction fragments.

Each enzyme cuts at the same place relative to its recognition sequence

restriction enzymes where identified initially in

bacteria

blunt ends from restriction enzymes

cuts are straight through both DNA strands at the line of symmetry

sticky ends from restriction enzymes

cuts are displaced equally on either side of line of symmetry

ends have either 5’ overhangs or 3’ overhangs

different restriction enzymes produce fragments of

different length

general formula for fragment length is

4^n (n=number of bases in the recognition site) with the assumption that the four bases are randomly distributed in the genome

in the discovery of restriction enzymes: the bacterium prevents phage replication (restriction) which happens because

the bacterial restriction endonucleases digest viral DNA into fragments, restricting the biological activity of the virus —> restriction enzymes

restriction and modification together are called

restriction-modification systems - a prokaryotic “immune system” against invaders

some experiments require random cutting of the DNA, meaning that

mechanical forces break phosphodiester bonds randomly and the resulting ends can be blunt or may have protruding single-stranded regions

molecular cloning

isolate a specific DNA fragment from all other fragments, making many identical copies

two basic steps of molecular cloning

1. insert DNA fragments into cloning vectors that ensure transport, replication, and purification of DNA inserts

2. insert recombinant DNA into living cells to be copied

three main features of a cloning vector (DNA fragments by themselves cannot replicate so they have to be inserted into a cloning vector)

1. an origin of replication

2. a selectable marker gene (antibiotic resistance)

3. a polylinker = region with several restriction enzyme sites

vector and DNA fragment (insert) digested with the same restriction enzyme generates

complementary sticky ends even if DNA comes from different organisms (bacterial and human)

DNA ligase

used to seal the phosphodiester backbones between vector and fragment

plasmids

double-stranded DNA

replicate in bacterial cells

independent of the chromosome

usually small inserts (<20 kb)

artificial chromosomes

larger

bacterial artificial chromosomes (BACs), inserts up to 300 kb

yeast artificial chromosomes (YACs), inserts up to 2 Mb

each genomic DNA fragment can form a

different recombinant molecule

transformation

the process by which a cell or organism takes up foreign DNA (chemical transformation or electroporation)

genomic library

long-lived collection of cellular clones, contains copies of every sequence in the whole genome inserted into a suitable vector

each colony on the agar plate contains a

different recombinant plasmid each with a part of the human genome (or the genome of interest)

a “perfect” genomic library has

one copy of every sequence in the entire genome

genomic equivalent

number of clones in a perfect library

= length of genome / average size of inserts

problems with cloning

never 100% efficient

some fragments can be cloned more than once

in reality we need 4-5 genomic equivalents

sanger developed a method to sequence DNA based on knowledge of how

DNA replicates in cells

sanger sequencing generates a series of

single-stranded DNA fragments

the size of a genome/the number of genes do not reflect the

complexity of an organism

sequencing is challenging especially for large genomes

whole-genome shotgun sequencing (Celera)

1. create a genomic library of overlapping fragments (500-1000 bp) in plasmid vectors

2. sequence randomly chosen plasmids (“shotgun”)

3. assemble sequences into contigs - continuous base pair sequences

4. stop sequencing when the 24 chromosomes are covered

the whole-genome shotgun strategy for genome sequencing is a problem because of

transposable elements

thousands of copies and each repeat copy is longer than a sequence read

one cannot determine (human or computer) which unique sequences flanking a repeat belong together

paired-end sequencing

to go around the problems with whole-genome shotgun strategy

a BAC clone library

2 sequence reads for each insert, one from each of 2 primers

some paired reads will include unique sequences on both sides of a repeat

annotation

the process of determining which sequences do which tasks

a key aspect of the Human Genome Project

a way to find protein-coding exons is by

looking for open reading-frames (ORFs) (a reading-frame uninterrupted by stop codons)

DNA can be read in six reading frames, three from each strand due to codone length

if a reading frame is >21 triplets, it is likely it encodes a

real protein

Darwin proposed a

descent with modification

the evolution of species from (extinct) ancestors

how do we trace changes in a genome?

finding conserved sequences

calculate the probability that another 50 bp DNA is 100% identical by chance

probability is obtained by raising 0.25 to the 50th power

a DNA sequence is a homolog of another DNA sequence if the

two derived from a common ancestor

when homology is present across species

the sequence is conserved

RNA is difficult to _________ and some RNAs are

sequence, low-abundance

cDNA (complementary DNA)

we can copy RNA into DNA then sequence that DNA

converting RNA transcripts to cDNA process:

1. isolate RNA from the cell

2. mRNAs sorted from rest of RNA based on poly A tails at 3’ end of mRNA

3. in vitro synthesis (DNA synthesis is primed, polymerization of first cDNA strand from 3’ end of the mRNA)

4. mRNA is digested with RNase (3’ end of cDNA folds back and acts as a primer for 2nd strand synthesis)

5. in the presence of dNTPs and DNA polymerase, the first cDNA strand acts as a template to synthesize the second cDNA strand

cDNA library includes only ____ and tells us?

includes only exons

tells us what was transcribed in that cell

alternative splicing means a single gene can ____________________ which?

a single gene can produce different proteins

complicates the prediction of the proteome

important to sequence many individual cDNA clones from libraries made using mRNAs from different tissues

the part of the genome corresponding to exons is the

exome (1.5-2% of the genome)

the increase in genome size correlates with the increase of

noncoding and repetitive sequences in more complex multicellular organisms

an increase in the number of epigenetic mechanisms

two types of particular DNA sequences found many times in genome

multicopy tandem repeats (CAG repeats)

transposable elements (sequences that can move around the genome)

most repetitive DNA does not have

known functions

repetitive DNA has important functions in several contexts:

telomeres - prevent the shortening of chromosomal ends

centromeres - bind proteins that help chromosomal sorting in mitosis/meiosis

gene deserts

regions of the genome that contain few or no genes

exons often encode protein domains which are

sequence of amino acids that fold into functional units

exon shuffling

generates new genes and new proteins

mediated by transposons

during meiotic crossover

gene families

groups genes closely related in sequence and function

abundant through genomes

gene families evolved by

duplication and divergence

duplicated DNA sequence products start out identical but eventualy

diverge via acucmulation of mutations

orthologous genes

arose from the same gene in the common ancestor

usually retain the same function

paralogous genes

arise by duplication

often refers to members of a gene family

pseudogenes

sequences that look alike, but do not function as, genes

rapidly accumulate mutations faster than coding or regulatory sequences

eventually it may not be possible to recognize the gene they derives from

de novo genes

genes without homologs

many genes have no homologs/only have homologs in closely related species

the more we go back in time, the more

chromosomal rearrangements

the average size of syntenic blocks gets smaller with increasing

evolutionary distance

combinatorial amplification

combining a set of basic elements in many different ways

combinatorial amplification at the DNA level results in

greater complexity from fewer genes

combinatorial strategies at the RNA level may lead to 

gene amplification and diversity

problems with the reference human genomes

the reference genome is not a “healthy” genome and is also not the most common genome

individuals who provided DNA may have been carriers of disease alleles

the 1,000 genomes project reveals that there is a

high degree of copy number variation and structure variation among healthy individuals

what were the 8 isolates that was sequenced from the streptococcus group B 

a “core genome” - ~80% of each genome

a “variable genome” - not present in all strains

the order of hemoglobin genes on the chromosomes reflects the

timing of expression

chromosomal organization

genes placed in the order of their expression during development

genes oriented in same direction on the chromosome

locus control region (LCR)

controls the sequential gene expression by interacting with enhancers and transcription factors

hereditary persistence of fetal hemoglobin

rare condition

should be lethal

deletion of delta and beta genes

gamma genes continue to be expressed during adulthood

results in near normal level of health

hemolytic anemias

some mutations change amino acids in alpha or beta-globin and change their 3D structure

causes destruction of RBCs

examples: sickle cell anemia, other anemias

thalassemias

mutations reduce/eliminate production of one of the two globin polypeptides

associated with alpha-globin deletions

a range of phenotypes

alpha-globin

required during fetal and adult development

alpha-thalassemias detrimental in utero

beta-globin

required after birth

beta-thalassemias detrimental after birth

transgenic organism

has a gene from another individual in the same species/a different species

transgene

the gene introduced into a transgenic organism

several strategies to introduce the DNA

inject the DNA into the cell

a viral particle carries the DNA into the cell

temporarily disrupt the plasma membrane

transgenes can be integrated into

the chromosome, the plasmid

if transgenes are transmitted across generations it has to be integrated into

gametes

a fertilized egg contains 2

pronuclei (maternal pronucleus and paternal pronucleus)

pronuclear injection is used to create a (and what is the process)

transgenic mouse (fertilized eggs harvested from the female mouse, linear DNA (transgene) is injected into one of the pronuclei, fertilized egg inserted into the reproductive tract of pseudo-pregnant mice)

25-50% of the time, integration is into a

random genomic location

if insertion of transgene happened before the first mitotic division,

every cell has the insert

if insertion of the transgene happened after the first cell division, the transgene will be in

some cells and not others (mosaic)

mice are bred to produce stable

transgenic lines

the P element (and how are transgenic flies made using P element transformation)

a type of transposable element in Drosophila

used as a vector for gene transfer

contains a transgene and a visible marker between inverted repeats

a helper plasmid contains the transposase gene

the two plasmids are injected into embryos

transgenic flies are recognized because they have red eyes

the resulting adult flies are mosaic

the visible marker helps identify transgenic flies in subsequent crosses

postranslational modifications are absent/present in

absent in bacteria

present in mammalian cells

gaucher disease

autosomal recessive

glucocerebrosidase deficiency in lysosomes

glucosylceramide accumulates in cells

the first plant-made drug approved by the FDA

glucocerebrosidase

pharming

farming and pharmaceutical

the use of transgenic animals/plants to produce protein drugs

blood coagulation

thrombin cleaves fibrinogen

fibrinogen helps form a blood clot

antithrombin 3

prevents thrombin inadvertent activation

atryn

blood factor antithrombin III

produced in goats, used to treat blood clots

antithrombin III deficiency

congenital disease

autosomal dominant but can also be recessive

predisposition to blood clots

reproductive cloning

the genome of a somatic cell becomes the genome of every somatic cell in a different individual

somatic cell nuclear transfer (SCNT)

used to create reproductive clones

a diploid nucleus of a somatic cell from one individual inserted into an egg cell whose nucleus has been removed

EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) is needed by what to make what

plants need it to make aromatic amino acids