1. Alu Human Polymorphism
(Differences between individuals at chromosomal regions)
Polymorphic means “many forms”
Many polymorphisms are located in the non-coding DNA.

2. 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?

3. 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)
140,000,000
A. thaliana (mustard plant)
24,000
120,000,000
Mammalian
20,000-25,000
3,000,000,000
Is the number of genes surprising?
What can we say about the relationship between genome size and number of genes?
What can we say about number of genes and the complexity of the organism?
What could account for the small number of mammalian genes but the large amount of complexity?

4. Human Genome What is a gene? How much of the genome are “genes”?
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?

6. 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)

7. 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.

8. Alu elements http://www.geneticorigins.org/pv92/aluframeset.htm
Under media/animations
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

9. 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

10. 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

11. 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

12. 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

13. 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)

14. Human Genome Online resources for mapping genetic diseases

15. 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

16. 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: 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

17. The primer/loading dye is sucrose and cresol red
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.

18. Alu Lab Possible problems Lose cell pellet at the beginning
No amplification
Maybe didn’t get complete lysis

19. 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
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

20. 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

21. Alu within the Alu Alu sequence (non-mutated):
ggccgggcgcggtggctcacgcctgtaatcccagcactttgggaggccgaggcgggcggatcacgaggtcaggagat gagaccatcccggctaaaacgctgaaacctcgtctctactaaaaatacaaaaaattagccgggcgtagtggcgggcgctgtagtcccagctacttgggaggctgaggcaggagaatggcgtgaacccgggaggcggagcttgcagtgagccgagatcctgccactgcactccagcctgggcgacagagcgagactccgtctcaaaaaaaaaaaaaaaaaaaaaaaaaaa

22. Alu within the Alu

24. Alu within the Alu: sequence results
Full Alu sequence
½ Alu sequence

25. Website
National Geographic articles on using the Y-chromosome to trace migration out of Africa
DNA interactive website: links to Human Identification, Recovering the Romanovs, Human Origins and Genes and Medicine