Studying differences/similarities in Individuals

1. Studying differences/similarities in Individuals Methods used to study differences between individuals RFLP SNP DNA Repeats

2. Genetic polymorphism • Genetic Polymorphism: A difference in DNA sequence among • individuals, groups, or populations. • Genetic mutations are a kind of genetic polymorphism. Genetic Variation Single nucleotide Polymorphism (point mutation) Repeat heterogeneity

3. Repeats and DNA fingerprint Variation between people- small DNA change – a single nucleotide polymorphism [SNP] – in a target site, RFLPs and point mutations are proof of variation at the DNA level. Satellite sequences: a short sequence of DNA repeated many times. Chr1 Interspersed Chr2 tandem

4. E E E E E E E E Repeats Micro-satellite Extremely small repeats (2-10bp)- GCGCGCGC AGCAGCAGCAGC Mini-satellite- larger repeats (20-100 bp long) Chr2 3 1 4 0.5 Repeats can be dispersed or in tandem- tandem 2 5 6 Chr1

5. E E E E E E E E Mini Satellite Repeats and Blots Mini Satellite sequences: a short sequence (20-100bp long) of DNA repeated many times 2 5 6 Chr1 Chr2 3 1 4 0.5 tandem 5 Take Genomic DNA Digest with EcoRI Probe southern blot with repeat probe 3 1

6. E E E E Repeat expansion/contraction Tandem repeats expand and contract during recombination. Mistakes in pairing leads to changes in tandem repeat numbers These can be detected by Southern blotting because as the number of repeats expand at a specific site, the restriction fragment at that site expands in size An allele of a mini-satellite varies by the number of repeats One repeat to many repeats- (varying in length from 0.5 to 20 kb) Chr1 Individual 1 2 Chr1 Individual 2 3 Ind1 Ind2 5 There are on average between 2 and 10 alleles (repeats) per mini-sat locus 3 1

7. E E E E Homozygous/heterozygous? Ind1 Ind2 Ind3 Chr1 Chr1 5 3 1

8. Micro-satellite and PCR Microsatellite repeat expansion and contraction is investigated using PCR and gels instead of gels and southern blots AGCGTCAGCGCGCGCTTATTGA TCGCAGTCGCGCGCGAATAACT AGCGTCAAATAACT 22 bp PCR fragment AGCGTCAGCGCGCGCGCGCGCTTATTGA TCGCAGTCGCGCGCGCGCGCGAATAACT AGCGTCAAATAACT 28 bp PCR fragment

9. DNA fingerprint 1 2 3 4 1 2 The use of microsatellite analysis in genetic profiling. In this example, 2 totally different microsatellites(1) and (2) located on the short arm of chromosome 6 have been amplified by the polymerase chain reaction (PCR). The PCR products are labeled with a blue or green fluorescent marker and run in a gel each lane showing the genetic profile of a different individual. Each individual has a different genetic profile because each person has a different number of microsatellite length repeats, the number of repeats giving rise to bands of different sizes after PCR. Locus1 locus2 Individual1 2,4 6,6 Individual2 2,6 1,4 Individual3 5,6 4,5

10. FBI and Microsatellite The FBI uses a set of 13 different microsatellite markers in forensic analysis. 13 sets of specific PCR primers are used to determine the allele present in the test sample for each marker. The marker used, the number of alleles at each marker and the probability of obtaining a random match for a marker is shown. How often would you expect an individual to be mis-identified if all 13 markers are analyzed Locus No. of alleles probability of random match A 11 0.112 B 19 0.036 C 7 0.081 D 7 0.195 E 10 0.062 F 10 0.075 G 10 0.158 H 11 0.065 I 10 0.067 J 8 0.085 K 8 0.089 L 15 0.028 M 20 0.039 P= 0.112x0.036x0.081x0.195x----- = = 1.7x10-15

11. DNA finger printing Variation between people- small DNA change – a single nucleotide polymorphism [SNP] – in a target site, RFLPs and SNPs are proof of variation at the DNA level, Satellite sequences: a short sequence of DNA repeated many times. Micro satellite are 2-4 bp repeats in tandem repeats 15-100 times in a row Mini satellite are 20-100 bp repeats in tandem (0.5 to 20kb long) Class size No of loci method Micro ~200bp 200,000 PCR Mini 0.2-20kb 30,000 southern blot SNP 1 bp 100 million PCR/microarray

12. The innocence project

13. Commits serious crime Not in database Commits minor crime DNA in database DNA from crime scene has Partial match. Focus on family. Prop 69 2004

14. Jeffreys In 1986, the Enderby murder case, a case local to Leicester, saw the first use of DNA profiling in criminology. Two young girls had been raped and murdered, one in 1983 and one in 1986. After the second murder, a young man was arrested and gave a full confession. The police thought he must have committed the first murder as well, so they asked Professor Jeffreys to analyse forensic samples – semen from the first and second victims, samples from the victims, and blood from the prime suspect. "The police were right – both girls had been raped by the same man," says Professor Jeffreys. "But it wasn't the man who had confessed. At first I thought there was something wrong with the technology, but we and the Home Office's Forensic Science Service did additional testing and it was clear that it was not his semen. He had given a false confession and was released – so the first time DNA profiling was used in criminology, it was to prove innocence." Blood samples from more than 5000 men in the local community were collected. The murderer nearly got away with it – sending a proxy in to give a blood sample – but eventually he was apprehended and got two life sentences.

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16. Individuals Methods used to study differences between individuals DNA Repeats RFLP SNP

17. Analysis of globin gene (deletion) M M H M H 0.2kb 1.1kb Exon1 Exon2 D 0.3kb deletion MstII HindIII Marker WT Marker Del WT Del 1.1 1.35 1 1.05 0.2

18. Very small Deletion (of a restriction site) E E E E E E 1kb 2kb 3kb 4kb 5kb 4.5kb 0.5kb 8kb 9kb GeneR GeneC GeneX GeneA H H 3kb Marker Marker Marker Marker EcoRI EcoRI WT Deletion

19. RFLP analysis RFLP= Restriction fragment length polymorphism Refers to variation (presence or absence) in restriction sites between individuals Because of mutations in Restriction sites These are extremely useful and valuable for geneticists (and lawyers) On average two individuals (humans) vary at 1bp in every 300-1000 bp The human genome is 3x109 bp This means that they will differ in more than 3 million bp!!! By chance these changes will create or destroy the recognition sites for restriction enzymes

20. RFLP Lets generate a EcoRI map for the region in one individual 3kb 4kb The the same region of a second individual may appear as GAATTC GAATTC GAATTC 7kb Marker 1 2 GAATTC GAGTTC GAATTC Normal GAATTC Mutant GAGTTC EcoRI

21. RFLP The internal EcoRI site is missing in the second individual For X1 the sequence at this site is GAATTC CTTAAG This is the sequence recognized by EcoRI The equivalent site in the X2 individual is different GAGTTC CTCAAG This sequence IS NOT recognized by EcoRI and is therefore not cut Now if we examine a large number of humans at this site we may find that 25% possess the EcoRI site and 75% lack this site. We can say that a restriction fragment length polymorphism exits in this region These polymorphisms usually do not have any phenotypic consequences Silent mutations that do not alter the protein sequence because of redundancy in codon usage, localization in introns or non-genic regions

22. RFLP RFLP are identified by southern blots In the region of the human X chromosome, two forms of the X-chromosome are Segregating in the population. X1 B R R R B R 4 5 3 6 3.5 1 2 Digest DNA with EcoRI or BamHI and probe with Probe1/ probe2 What do we get? X2 B R R B R 8 4 6 3.5 1 2

23. RFLP in individuals If we used probe1 for southern blots with a BamHI digest what would be the results for X1/X1, X1/X2 and X2/X2 individuals? X1/X1 X1/X2 X2/X2 Probe1 BamHI 18 18 18 If we used probe1 for southern blots with a EcoRI digest what would be the results for X1/X1, X1/X2 and X2/X2 individuals? X1/X1 X1/X2 X2/X2 Probe1 EcoRI 5, 3 8, 5, 3 8 Probe2 EcoRI 9.5 9.5 9.5 X1 X2 4 4 5 8 3 6 6 3.5 3.5 B B R R R R R R B B R R 1 1 2 2

24. RFLP RFLP’s are found by trial and error They require an appropriate probe and appropriate enzyme They are very valuable because they can be used just like any other genetic marker to map genes They are employed in recombination analysis (mapping) in the same way as conventional morphological allele markers are employed The presence of a specific restriction site at a specific locus on one chromosome and its absence at a specific locus on another chromosome can be viewed as two allelic forms of a gene The phenotype in this case is a Southern blot rather than white eye/red eye 4 6 2 2 R R R R 1 X1 1 4 5 R R R 2 4 8 2 X2 1 2 4 3 R R R R R R R 1 2

25. X1 1 4 5 R R R 4 6 2 2 2 R R R R 1 X2 1 2 4 3 R R R R 2 Probe1 Probe2 6+2 8 X1/X1 individual 5 3 X2/X2 individual 4 8 2 R R R 1

26. Using RFLPs to map human disease genes 8 EcoRI Probe1 6 5 9 EcoRI Probe2 EcoRI Probe3 3 1 1 2 3 Which RFLP pattern segregates specifically with all of the diseased individual BUT NOT WITH NORMAL INDIVIDUALS? 1, 2 or 3 Which band segregates with the phenotype Top or bottom? Using DNA probes for different RFLPs you can screen individuals for a RFLP pattern that showsco-inheritance with the disease Conclusion: the actual mutation resides at or near RFLP1 bottom band

27. RFLPs and Mapping unknown Genes Lets review standard mapping: To map any two genes with respect to one another, they must be heterozygous at both loci.Gene W and B are responsible for wing and bristle development W B Telomere Centromere To find the map distance between these two genes we need allelic variants at each locus W=wings B=Bristles w= No wings b= no bristles To measure genetic distance between these two genes, the double heterozygote is crossed to the double homozygote

28. Mapping To map a gene with respect to another, you perform crosses and measure recombination frequency between the two genes. Gene W and B are responsible for wing and bristle development W B Telomere Centromere To find the map distance between these two genes we need ALLELIC variants at each locus W=wings B=Bristles w= No wings b= no bristles To measure distance between these two genes, the double heterozygote is crossed to the double homozygote wb/wb Males No wings No bristles WB/wb Female Wings Bristles X ----W--------B--- ----w--------b--- ----w--------b--- ----w--------b---

29. Mapping Male gamete (wb) Genotype phenotype WB WB/wb Wings Bristles 51 wb/wb No wings No bristles 43 Wb/wb Wings No bristles 3 wB/wb No wings Bristles 4 wb Wb Female gamete wB Map distance= # recombinants /Total progeny 7/101= 7 M.U.

30. B 3 2 E E E b E 5 E Mapping Both the normal and mutant alleles of gene B (B and b) are sequenced and we find W B Telomere Centromere GAATTC AAATTC The mutation disrupts the sequence and alters a EcoRI site! If DNA is isolated from B/B, B/b and b/b individuals, cut with EcoRI and probed in A Southern blot, the pattern that we will obtain is B/B Bristle B/b Bristle b/b No bristle

31. Mapping To find the map distance between two genes we need ALLELIC variants at each locus Therefore in the cross (WB/wb x wb/wb), the genotype at the B locus can be distinguished either by the presence and absence of bristles OR by a Southern blot WB/wb x wb/wb Female Male WingsNo wings BristlesNo Bristles Southern blot: Southern blot: 5 and 2 kb band5 kb band You can use RFLPs instead of genes as markers along a chromosome Just like Genes, RFLPs mark specific positions on chromosomes and can be used for mapping.

32. Mapping Male gamete (wb) Genotype phenotype Parental WB WB/wb Wings 51 5kb 2kb wb wb/wb No wings 43 5kb Recombinant Wb Wb/wb Wings 3 5kb wB wB/wb No wings 4 5kb 2kb Female gamete Map distance= # recombinants /Total progeny 7/101= 7 M.U.

33. W B R Centromere Telomere Mapping To find the map distance between genes, multiple alleles are required. We know the distance between W and B by the classical method because multiple alleles exist at each locus (W & w, B & b). It is 7MU. We know the distance between B and R by the classical method as 20MU. 7MU 20MU C Now suppose you find a new gene C. You could map this gene with respect to Genes W, B and R using classical methods. However, what if it is difficult to study the function of this new gene (the phenotype is difficult to see with the naked eye) If the researcher identifies an RFLP in this gene you can map the gene mutation by simply following the RFLP.

34. C c 6 2 E E E E 8 E Mapping With this RFLP, the C gene can be mapped with respect to other genes in any cross. Genotype/phenotype relationships for the W and C genes WW and Ww = Red eyes ww = white eyes CC = 8kb band C/c = 8, 6, 2 kb bands cc = 6, 2 kb bands To determine map distance between R and C, the following cross is performed W C w c ------------ ------------ ------------ ------------ w c w c

35. Mapping 7MU 20MU C W B R w c(6,2) W C(8) w w c(6,2) c(6,2) Male gamete (wc) Genotype phenotype Parental WC WC/wc Red/8,6,2 wc wc/wc white/6,2 Recombinant Wc Wc/wc Red/6,2 wC wC/wc white/8,6,2 45 45 Female gamete 5 5 Map distance between W and C is 10MU

36. W or w B or b RFLP1 Probe1 7kb or 4kb RFLP2 Probe2 1kb or 2kb RFLP3 Probe3 3kb or 9kb R or r Centromere Centromere Telomere Telomere Mapping Prior to RFLP analysis, only a few classical markers existed in humans (approximately 200) Now over 7000 RFLPs have been mapped in the human genome. Newly inherited disorders are now mapped by determining whether they are linked to previously identified RFLPs 7MU 20MU

37. Individuals Methods used to study differences between individuals RFLP SNP DNA Repeats

38. Genetic polymorphism • Genetic Polymorphism: A difference in DNA sequence among • individuals, groups, or populations. • Genetic Mutation: A change in the nucleotide sequence of a • DNA molecule. • Genetic mutations are a subset of genetic polymorphism Genetic Variation Single nucleotide Polymorphism (point mutation) Repeat heterogeneity

39. SNP • A Single Nucleotide Polymorphism is a source variance in a genome. • A SNP ("snip") is a single base change in DNA. • SNPs are the most simple form and most common source of • genetic polymorphism in the human genome (90% of all human DNA polymorphisms). • There are two types of nucleotide base substitutions resulting in SNPs: • Transition: substitution between purines (A, G) or between pyrimidines (C, T). Constitute two thirds of all SNPs. • Transversion: substitution between a purine and a pyrimidine. • While a single base can change to all of the other three bases, most SNPs have only one allele.

40. SNPs- Single Nucleotide Polymorphisms -----------------------ACGGCTAA -----------------------ATGGCTAA Instead of using restriction enzymes, these are found by direct sequencing/PCR They are extremely useful for mapping Markers Classical Mendelian ~200 RFLPs 7000 SNPs 1.4x106 SNPs occur every 300-1000 bp along the 3 billion long human genome Many SNPs have no effect on cell function Note: RFLPs are a subclass of SNPs

41. SNPs Humans are genetically >99 per cent identical: it is the tiny percentage that is different Much of our genetic variation is caused by single-nucleotide differences in our DNA : these are called single nucleotide polymorphisms, or SNPs. As a result, each of us has a unique genotype that typically differs in about three million nucleotides from every other person. SNPs occur about once every 300-1000 base pairs in the genome, and the frequency of a particular polymorphism tends to remain stable in the population. Because only about 3 to 5 percent of a person's DNA sequence codes for the production of proteins, most SNPs are found outside of "coding sequences".

42. How did SNPs arise? F2a----ACGGACTGAC----CCTTACGTTG----TACTACGCAT---- | F1 ----ACTGACTGAC----CCTTACGTTG----TACTACGCAT---- P ----ACTGACTGAC----CCTTACGTTG----TACTACGCAT---- | F1 ----ACTGACTGAC----CCTTACGTTG----TACTAGGCAT---- | | F2b----ACTGACTGAC----CCATACGTTG----TACTAGGCAT---- Compare the two F2 progeny Haplotype1 (F2a) = SNP allele1 ----ACGGACTGAC----CCTTACGTTG----TACTACGCAT---- Haplotype2 (F2b) = SNP allele2 ----ACTGACTGAC----CCATACGTTG----TACTAGGCAT----

43. Each of 1013 cells in the human body receives approximately thousand DNA lesions per day (Lindahl and Barnes 2000) When these mutations are not repaired they are fixed in the genome of that particular cell If a mutation is fixed in germ cells that go on to be fertilized and form an embryo they will be propagated to progeny

44. SNPs, RFLPs, point mutations GAATTC GAATTC GAATTC GAATTC GAATTC GAATTC GAGTTC GAATTC GAATTC GACTTC RFLP SNP RFLP Pt mut SNP Pt mut SNP SNP

45. Coding Region SNPs • Types of coding region SNPs • Synonymous: the substitution causes no amino acid change to • the protein it produces. This is also called a silent mutation. • Non-Synonymous: the substitution results in an alteration of • the encoded amino acid. A missense mutation changes the • protein by causing a change of codon. A nonsense mutation • results in a misplaced termination. • More than half of all coding sequence SNPs result in • non-synonymous codon changes.

46. Alzheimer’s SNP Occasionally, a SNP may actually cause a disease. SNPs within a coding sequence are of particular interest to researchers because they are more likely to alter the biological function of a protein. One of the genes associated with Alzheimer's, apolipoprotein E or ApoE, is a good example of how SNPs affect disease development. This gene contains two SNPs that result in three possible alleles for this gene: E2, E3, and E4. Each allele differs by one DNA base, and the protein product of each gene differs by one amino acid. Each individual inherits one maternal copy of ApoE and one paternal copy of ApoE. Research has shown that an individual who inherits at least one E4 allele will have a greater chance of getting Alzheimer's. Apparently, the change of one amino acid in the E4 protein alters its structure and function enough to make disease development more likely. Inheriting the E2 allele, on the other hand, seems to indicate that an individual is less likely to develop Alzheimer's.

47. Intergenic SNPs Researchers have found that most SNPs are not responsible for a disease state because they are intergenic SNPs Instead, they serve as biological markers for pinpointing a disease on the human genome map, because they are usually located near a gene found to be associated with a certain disease. Scientists have long known that diseases caused by single genes and inherited according to the laws of Mendel are actually rare. Most common diseases, like diabetes, are caused by multiple genes. Finding all of these genes is a difficult task. Recently, there has been focus on the idea that all of the genes involved can be traced by using SNPs. By comparing the SNP patterns in affected and non-affected individuals—patients with diabetes and healthy controls, for example—scientists can catalogALL of the DNA sequence variations in affected Vs unaffected individuals to identify mutations that underlie susceptibility for diabetes GACTTC GAATTC GAGTTC GAATTC GAATTC RFLP SNP RFLP Pt mut SNP Pt mut SNP SNP

48. How do you identify SNPs in individuals- PCR PCR is quick sensitive and robust and is useful when dealing with small amounts of DNA, or where rapid and high-throughput screening is required. PCR: * The polymerase chain reaction involves many rounds of DNA synthesis. * All DNA synthesis reactions require a template, a primer, a enzyme and a supply of nucleotides. In the standard PCR, two primers flank the target for amplification and face inwards. DNA synthesis therefore proceeds across the region between the primers. PCR results in exponential amplification of the target sequence. How does it work? The reaction begins by heating up the DNA template to 94°C, which splits (denatures) the double strands into single strands. The sample is then cooled to about 54°C, which allows the primers to stick (anneal) to the template. When the sample is heated up again to 72°C, the polymerase enzyme uses the primers as starting points to copy the single strands. Special DNA polymerase that can withstand high temperatures is used The cycle of denaturation, primer annealing and primer extension is repeated over and over again (using a machine that automates the heating and cooling of the samples), each time producing more copies of the original template. During repeated rounds of these reactions, the number of newly synthesized DNA strands increases exponentially. After 25 to 30 cycles, the initial template DNA will have been copied several million-fold. Doubling occurs in every cycle of the PCR leading to exponential amplification of the target. After 25 cycles there are over 8 000 000 copies! The PCR is useful where the amount of starting material is limited or poorly preserved. Examples of PCR applications include cloning DNA from single cells, prenatal screening for mutations in early human embryos, and the forensic analysis of DNA sequences in samples such as fingerprints, blood stains, semen or hairs. The PCR is also very useful where many samples have to be processed in parallel. For example, the large-scale analysis of single nucleotide polymorphisms involves PCR-based techniques

49. If a region of DNA has already been sequenced in one individual, the sequence information can be used to isolate and amplify that sequence from other individuals DNA in a population. Individuals with mutations in p53 are at risk for colon cancer To determine if an individual had such a mutation, prior to PCR one would have to clone the gene from the individual of interest (construct a genomic library, screen the library, isolate the clone and sequence the gene). With PCR, the gene can be isolated directly from DNA isolated from that individual. No lengthy cloning procedure necessary Only small amounts of genomic DNA required 30 rounds of amplification can give you >109 copies of a gene PCR

50. Heat and add primers PCR Heat resistant DNA polymerase Heat and add primers + DNA pol