Transcript Alu Human Polymorphism

Alu Human Polymorphism
(Differences between individuals
at chromosomal regions)
Polymorphic means “many forms”
Many polymorphisms are located
in the non-coding DNA.
Human Genome
• How many chromosomes does each human cell
have?
– 22 pairs of autosomal chromosome and 1 pair of sex
chromosomes
• What is the size of the human genome?
– The human genome is made up of 3 billion base pairs
• How many genes does the Human Genome
code for?
Genome Comparison
Organism
# of Genes
Genome Size (bp)
E.coli
4,200
4,600,200
S. cervisiae (yeast)
6,000
12,000,000
C. elgans (nematode)
14,000
100,000,000
D. melanogaster (fruit fly)
14,000
140,000,000
A. thaliana (mustard plant)
24,000
120,000,000
20,000-25,000
3,000,000,000
Mammalian
Human Genome
• What is a gene?
– A gene is segment of a genome that encodes
a single specific end product (a protein or
RNA) or a functional unit or information
• How much of the genome are “genes”?
– ~1.5% of the genome
• What accounts for the rest of the DNA in
the genome?
Human Genome
• Other items in genome
– Introns
– Repeated sequences (telomeres,
centromeres)
– Non-coding RNAs
– Transposons (SINES and LINES)
(Short Interspersed Elements: less than 500 bp
long and present 500,000 or more in the
genome and Long Interspersed Elements)
– Psuedogenes (genes that no longer produce
functional products)
Transposons –
Transposable Element
• Alu is a transposable element.
• A member of SINEs. 300 nucleotides.
• Name comes from a endonuclease AluI
that is found in the middle.
• Sometimes called “jumping genes”
but not really a jumping gene because it
doesn’t code for a protein.
Retrotransposon L1 (a LINE) supplies the rt
(reverse transcriptase) needed to jump.
Alu elements
• http://www.geneticorigins.org/pv92/aluframeset.htm
• Under media/animations
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Illustrations about the Alu element listed as How Alu jumps
Pedigree on Alu inheritance listed as PV92 inheritance
Theory: PV92 locus on Chromosome 16;
Homozygous ++; Heterozygous + -; Homozygous – (inherited in
Mendelian fashion)
– 731 bp with; 416 bp without
– Readily separated by gel electrophoresis
Alu elements
• Alu is a “defective” transposon of the
SINE (Short INterspersed Element) family
• Alu is about 300 nucleotides (base pairs)
in length
• Human chromosomes have more than 1
million Alu copies (10% of the genome by
mass)
• There is no evidence that an Alu can be
lost from a site – it can only be inserted
Alu elements
• Alu has the necessary sequences to be
transcribed by RNA Pol III
• The LINE (Long INterspersed Element) L1
supplies the reverse transcriptase needed
for Alu to jump
– L1 is a defective retrovirus
– It nicks the DNA close to the sequence
AATTTT
– The AAAA of Alu transcript binds to the TTTT
of the nicked DNA – the acts as a primer for
the L1 to bind to
Alu Elements
• What are the consequences and benefits
of having an Alu element?
• Alu inserting into exons has been
implicated in neurofibromatosis, cancer
and heart attacks
• They can provide alternate splicing in 5%
of genes which leads to genetic diversity
• Can provide information on migration and
evolution
Alu elements
• Alu elements are only found in the primate
branch
• Each Alu insertion is a unique event and is
inherited from each parent
– Most occurred millions of years ago and are often on
both pairs of chromosomes
– There are Alu elements that have occurred since
humans branched from other primates
– This gives rise to dimorphic Alus from the last
hundreds of thousands of years
– Estimated to jump 1:200 live human births
Alu Elements
• The Alu element that we will be looking at
in on chromosome 16
• There is no phenotypic data associated
with this locus (i.e. does not associate with
disease, trait, or sex determination)
Human Genome
Online resources for mapping genetic
diseases
• http://gslc.genetics.utah.edu/units/disorder
s/sloozeworm/scenario.cfm
• http://gslc.genetics.utah.edu/units/disorder
s/pedigree/mapgene.cfm
Alu Lab
• What is the benefit of saline? Saline is a very reproducible
way of harvesting cells – it loosens a large number of single cheek
cells
• What about food contamination?
• Don’t eat before lab; Brush your teeth, etc. after you
• Have breakfast. If molecules of food get in, it’s ok.
– No effect because the element is only found in primates
• Why are we boiling the samples?
– To lyse open the cells and release the DNA
• What is Chelex?
– Chelex is a metal binding resin
– It is used to scavenge metal that can contaminate PCR reactions
Alu Lab
• Where do the primers amplify?
– Primers amplify region on chromosome 16 – outside of the Alu
region – the primers do not amplify Alu itself
• What will the products (+/+, +/- and -/-) look like?
– -/- has one smaller band around 400bp, +/+ has one larger band
around 700bp; +/- has two bands one at 400 and one at 700
– There is a picture of a control gel on:
http://www.geneticorigins.org/pv92/aluframeset.htm under the
theory section
• Why is there no control for the PCR reaction?
– There is no need for a control because all samples will produce
a band therefore it will tell us if the DNA was good without a
need for an additional control
• The primer/loading dye is sucrose and
cresol red. Allows it to be loaded in the gel
wells and stay there and see where it is.
• The Ready-to-Go PCR bead contains
reagents: Taq DNA polymerase, Tris-HCl
(ph 9.0); KCl; MgCl2; and dNTPs
• Plasmid pBR322 is digested with
restriction endonuclease BstNI – produces
markers. (This will be the ladder). It
produces fragments at 1857 bp; 1058 bp;
929 bp; 383 bp; and 121 bp.
Alu Lab
Possible problems
– Lose cell pellet at the beginning
– No amplification
• Maybe didn’t get complete lysis
Alu within the Alu
Alu paper is on the wiki listed under Human DNA Polymorphism: Recent
Insertion of an Alu element within a Alu insertion
Online alignment tool
• http://www.ch.embnet.org/software/LALIGN_form.html
• To use Alignment tool copy the mutated large Alu sequence into the box
labeled 1st query sequence
– Make sure to name the sequence in box labeled First sequence title (I
named this Mut)
• Paste Alu element (non-mutated) sequence into box labeled 2nd
sequence query
– Make sure to name the sequence in box labeled Second sequence title (I
named this Alu)
• Click Run Align at the bottom of the page
• This will produce the alignment with the details about what bases were
aligned and the % of identity
– This will give you three alignments with the Alu element
Alu within the Alu
Mutated larger Alu sequence (from GenBank extension
number AF302689):
aactgggaaa atttgaagag aaagtcacac agatacattt cagtaaggtt gtctctgtta
61 cttgaggctt acaagaagga aagaaggccg ggcgcggtgg ctcacgcctg taatcccagc
121 actttgggag gccgaggcgg gcggatcacg aggtcaggag atcgagacca tcccggctaa
181 aacgctgaaa cctcgtctct actaaaaata caaaaaattg ccgggcgcgg tggctcacgc
241 ctgtaatccc agcactttgg gaggccgagg cgggtggatc atgaggtcag gagatcgaga
301 ccatcctggc taacaaggtg aaaccccgtc tctactaaaa atacaaaaaa ttagccgggc
361 gcggtggcgg ggcgcctgta gtcccagcta ctcgggaggc tgaggcagga gaatggcgtg
421 aacccgggaa gcggagcttg cagtgagccg agattgcgcc actgcagtcc gcagtccggc
481 ctgggcgaca gagcgagact ccgtctcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
541 aaaaaaaaaa aaaataccaa aaattagccg ggcgtagtgg cgggcgcctg tagtcccagc
601 tacttgggag gctgaggcag gagaatggcg tgaacccggg aggcggagct tgcagtgagc
661 cgagatcctg ccactgcact ccagcgtggg cgacagagcg agactccgtc tcaaaaaaaa
721 aaaaaaaaaa aaaaaaagaa agaattccct ctctaaacac actctaacac acaggagttg
781 agaactca
Alu within the Alu
Alu sequence (non-mutated):
ggccgggcgcggtggctcacgcctgtaatcccagcactttgggaggccgaggcgggcggatcacgaggtcaggagat
gagaccatcccggctaaaacgctgaaacctcgtctctactaaaaatacaaaaaattagccgggcgtagtggcgggc
gctgtagtcccagctacttgggaggctgaggcaggagaatggcgtgaacccgggaggcggagcttgcagtgagcc
gagatcctgccactgcactccagcctgggcgacagagcgagactccgtctcaaaaaaaaaaaaaaaaaaaaaaa
aaaa
Alu within the Alu
Alu within the Alu:
sequence results
½ Alu sequence
Full Alu sequence
½ Alu sequence
Website
National Geographic articles on using the Ychromosome to trace migration out of Africa
http://news.nationalgeographic.com/news/2002/12/1
212_021213_journeyofman.html
DNA interactive website: links to Human
Identification, Recovering the Romanovs, Human
Origins and Genes and Medicine
http://www.dnai.org/d/index.html