1. Chapter 11
Polymorphisms

2. Polymorphism
A DNA polymorphism is a sequence difference compared to a reference standard that is present in at least 1%–2% of a population.
Polymorphisms can be single bases or thousands of bases.
Polymorphisms may or may not have phenotypic effects

3. Polymorphic DNA Sequences
Polymorphisms are found throughout the genome.
If the location of a polymorphic sequence is known, it can serve as a landmark or marker for locating other genes or genetics regions.
Each polymorphic marker has different versions, or alleles.

4. Types of Polymorphic DNA Sequences
RFLP: restriction fragment length polymorphisms
VNTR: variable number tandem repeats (8 to >50 base pairs)
STR: short tandem repeats (1–8 base pairs)
SNP: single-nucleotide polymorphisms

5. Types of Polymorphisms
Single nucleotide polymorphisms (SNPs)
A Single Nucleotide Polymorphism, or SNP (pronounced "snip"), is a small genetic change, or variation, that can occur within a person's DNA sequence.
Common SNPs are defined as >1% in at least one population
Rare SNPs are hard to identify and validate
But, it is estimated that there are a large number per individual
Some areas more variable than others (HLA)

6. Restriction Fragment Length Polymorphisms
Restriction fragment sizes are altered by changes in or between enzyme recognition sites.
GTCCAGTCTAGC
GAATTC
GTGGCAAAGGCT
CAGGTCAGATCG
CTTAAG
CACCGTTTCCGA
GAA A TC CG
TGGC C
AGGCT
CTT T AG GC
ACCG G
TCCGA
Point mutations GA
AGCGA
ATTC
GTGGC
AAAGGCT CT
TCGCT
TAAG
CACCG
TTTCCGA
Insertion
Fragment insertion (or deletion)
GTTCTAGC
AAA
GGCT
GTGG
TCAGATCG
TTT
CCGA
CACC
AAAAAA
TTTTT T

7. RFLP
RFLP (often pronounced "rif lip”) is used to follow a particular sequence of DNA as it is passed on to other cells.
RFLPs can be used in paternity cases or criminal cases to determine the source of a DNA sample. RFLPs can be used determine the disease status of an individual. RFLPs can be used to measure recombination rates which can lead to a genetic map with the distance between RFLP loci measured in centiMorgans.

8. RFLP
An RFLP is a sequence of DNA that has a restriction site on each end with a "target" sequence in between.
A target sequence is any segment of DNA that bind to a probe by forming complementary base pairs.
A probe is a sequence of single-stranded DNA that has been tagged with radioactivity or an enzyme so that the probe can be detected. When a probe base pairs to its target, the investigator can detect this binding and know where the target sequence is since the probe is detectable.

9. RFLP
RFLP produces a series of bands when a Southern blot is performed with a particular combination of restriction enzyme and probe sequence

10. RFLP
Recall: EcoR I binds to its recognition sequence GAATTC and cuts the double-stranded DNA as shown
EcoRI
-GAATTC-
-CTTAAG-

11. In the segment of DNA shown below, you can see the elements of an RFLP; a target sequence flanked by a pair of restriction sites. When this segment of DNA is cut by EcoR I, three restriction fragments are produced, but only one contains the target sequence which can be bound by the complementary probe sequence (purple).

12. Restriction Fragment Length Polymorphisms
RFLP genotypes are inherited.
For each locus, one allele is inherited from each parent.
Southern blot
band patterns

13. Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-
Let's look at two people and the segments of DNA they carry that contain this RFLP (for clarity, we will only see one of the two stands of DNA). Since Jack and Jill are both diploid organisms, they have two copies of this RFLP. When we examine one copy from Jack and one copy from Jill, we see that they are identical:
Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-
Jill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-

14. When we examine their second copies of this RFLP, we see that they are not identical. Jack 2 lacks an EcoR I restriction site that Jill has 1.2 kb upstream of the target sequence (difference in italics).
Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC- Jill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC-

15. Therefore, when Jack and Jill have their DNA subject to RFLP analysis, they will have one band in common and one band that does not match the other's in molecular weight:

16. Parentage Testing by RFLP
Which alleged father’s genotype has the paternal alleles?
Child
Locus
AF 1
AF Mother

17. Evidence Testing by RFLP
Which suspect—S1 or S2—was at the crime scene?
(V = victim, E = crime scene evidence, M = molecular weight standard)
M S1 S2 V E M
Locus Locus Locus 3

18. Short Tandem Repeat Polymorphisms (STR)
STRs are repeats of nucleotide sequences.
AAAAAA…: mononucleotide
ATATAT…: dinucleotide
TAGTAGTAG…: trinucleotide
TAGTTAGTTAGT…: tetranucleotide
TAGGCTAGGCTAGGC…: pentanucleotide
Different alleles contain different numbers of repeats.
TTCTTCTTCTTC : four-repeat allele
TTCTTCTTCTTCTTC: five-repeat allele

19. Short Tandem Repeat Polymorphisms
STR alleles can be analyzed by fragment size (Southern blot).
CAAGATCG
CCGG
CACCG
TCGATCGATCGATCGA
CGAC
CACC
GTTCTAGC
GGCC
GTGGC
AGCTAGCTAGCTAGCT
GCTG
GTGG
One repeat unit
tandem repeat
Restriction site
Allele 1
Allele
M M
Allele 2
GTTCTAGC
GGCC
GTGGC
AGCTAGCTAGCT
GCTG
GTGG
CAAGATCG
CCGG
CACCG
TCGATCGATCGA
CGAC
CACC

20. Short Tandem Repeat Polymorphisms
STR alleles can also be analyzed by amplicon size (PCR).
(Genotype)
Primers

21. Short Tandem Repeat Polymorphisms
Allelic ladders are standards representing all alleles observed in a population.
(Allelic ladder)
11 repeats
5 repeats
Genotype: 7,9
Genotype: 6,8

22. Short Tandem Repeat Polymorphisms
Multiple loci are genotyped in the same reaction using multiplex PCR.
Allelic ladders must not overlap in the same reaction.

23. Short Tandem Repeat Polymorphisms by Multiplex PCR
FGA
TPOX
D8S1179
vWA
PentaE
D18S51
D2S11
THO1
D3S1358

24. Amelogenin Locus, HUMAMEL
The amelogenin locus is not an STR.
The HUMAMEL gene codes for amelogenin-like protein.
The gene is located at Xp22.1–22.3 and Y.
X allele = 212 bp
Y allele = 218 bp
Females (X, X): homozygous
Males (X, Y): heterozygous

25. Analysis of Short Tandem Repeat Polymorphisms by PCR
STR genotypes are analyzed using gel or capillary gel electrophoresis.
Genotype: 7,9
11 repeats
5 repeats
11 repeats repeats
(Allelic ladder)

26. STR-PCR STR genotypes are inherited.
One allele is inherited from each parent.
Child’s alleles
Mother’s alleles
Father’s alleles

27. Parentage Testing by STR-PCR
Which alleged father’s genotype has the paternal alleles?

28. Evidence Testing by STR-PCR
Which suspect—S1 or S2—was at the crime scene?
(V = victim, E = crime scene evidence, AL = allelic ladder)
AL S1 S2 V E M
Locus Locus Locus 3

29. Short Tandem Repeat Polymorphisms: Y-STR
The Y chromosome is inherited in a block without recombination.
STR on the Y chromosome are inherited paternally as a haplotype.
Y haplotypes are used for exclusion and paternal lineage analysis.

30. Short Tandem Repeat Polymorphisms: mini-STR
Mini-STRs are STRs on smaller amplicons.
Recommended for degraded specimens
Used to identify remains from mass graves and disaster areas

31. MHC
Major histocompatibility complex (MHC) is a cell surface molecule encoded by a large gene family in all vertebrates. MHC molecules mediate interactions of leukocytes. MHC determines compatibility of donors for organ transplant as well as one's susceptibility to an autoimmune disease via crossreacting immunization. In humans, MHC is also called human leukocyte antigen (HLA).
Protein molecules—either of the host's own phenotype or of other biologic entities—are continually synthesized and degraded in a cell. Occurring on the cell surface, each MHC molecule displays a molecular fraction, called an epitope. The presented antigen can be either self or nonself.
The MHC gene family is divided into three subgroups—class I, class II, and class III.
We will discuss these later…..

32. SNPs & Function: We know so little..
Majority are “silent”: No known functional change
Alter gene expression/regulation
Promoter/enhancer/silencer
mRNA stability
Small RNAs
Alter function of gene product
Change sequence of protein

33. Large and Small Scale Polymorphisms
Copy Number of Polymorphisms
Regional “repeat” of sequence
10s to 100s kb of sequence
Estimate of >10% of human genome
Multi-copy in many individuals
Duplicons
90-100% similarity for >1 kb
5-10% of genome (5% exons elsewhere)
Multi-copy (high N) in all individuals

34. Copy Number Variation Across the Genome Frequency of CNVs
Most are uncommon (<5%)
Familial vs Unrelated Studies
Associated with Disease
OPN1LW Red/green colorblind
CCL3L1 Reduced HIV Infection
CYP2A6 Altered nicotine metabolism
VKORC1 Warfarin metabolism

35. Engraftment Testing Using STR
Allogeneic bone marrow transplants are monitored using STR.
Autologous
transplant
Allogeneic
Recipient
receives his or her own purged cells.
receives donor
cells.
A recipient with donor marrow is a chimera.

36. Engraftment Testing Using STR
There are two parts to chimerism testing: pretransplant informative analysis and post-transplant engraftment analysis.
Recipient
Before transplant
Donor
After transplant
Complete (full Mixed Graft failure
chimerism) chimerism

37. Engraftment Testing Using STR: Informative Analysis
STR are scanned to find informative loci (donor alleles differ from recipient alleles).
Which loci are informative?
Locus:

38. Engraftment Testing Using STR: Informative Analysis
There are different degrees of informativity.
With the most informative loci, recipient bands or peaks do not overlap stutter in donor bands or peaks.
Stutter is a technical artifact of the PCR reaction in which a minor product of n-1 repeat units is produced.

39. Examples of Informative Loci (Type 5) [Thiede et al
Examples of Informative Loci (Type 5) [Thiede et al. Leukemia 2004;18:248]
Recipient
Donor
Stutter
Recipient
Donor

40. Examples of Non-informative Loci (Type 1)
Donor
Recipient

41. Engraftment Testing Using STR: Informative Analysis
Which loci are informative?
vWA TH Amel TPOX CSF1PO

42. Engraftment Testing Using STR: Engraftment Analysis
Using informative loci, peak areas are determined in fluorescence units or from densitometry scans of gel bands.
A(R) = area under recipient-specific peaks
A(D) = area under donor-specific peaks
A(D)
A(R)
A(R) + A(D)

43. Engraftment Testing Using STR: Engraftment Analysis
Formula for calculation of % recipient or % donor (no shared alleles).
% Recipient DNA =
A(R) + A(D)
A(R)
× 100
% Donor DNA =
A(R) + A(D)
A(D)
× 100

44. Engraftment Testing Using STR: Engraftment Analysis
Calculate % recipient DNA in post-1 and post-2:
Use the area under these peaks to calculate percentages.

45. Engraftment Analysis of Cellular Subsets
Cell subsets (T cells, granulocytes, NK cells, etc.) engraft with different kinetics.
Analysis of cellular subsets provides a more detailed description of the engrafting cell population.
Analysis of cellular subsets also increases the sensitivity of the engraftment assay.

46. Engraftment Analysis of Cellular Subsets
T cells (CD3), NK cells (CD56), granulocytes, myeloid cells (CD13, CD33), myelomonocytic cells (CD14), B cells (CD19), stem cells (CD34)
Methods
Flow cytometric sorting
Immunomagnetic cell sorting
Immunohistochemistry + XY FISH

47. Engraftment Analysis of Cellular Subsets
Detection of different levels of engraftment in cellular subsets is split chimerism.
R D T G
R = recipient alleles
D = donor alleles
T = T-cell subset (mostly recipient) G = granulocyte subset (mostly donor)

48. Single Nucleotide Polymorphisms (SNPs)
Single-nucleotide differences between DNA sequences.
One SNP occurs approximately every 1250 base pairs in human DNA.
SNPs are detected by sequencing, melt curve analysis, or other methods.
99% have no biological effect; 60,000 are within genes.

49. SNP Detection by Sequencing
T/T T/A A/A
5′ AGTCTG
5′ AG(T/A)CTG
5′ AGACTG

50. SNP Haplotypes
SNPs are inherited in blocks or haplotypes.

51. Applications of SNP Analysis
SNPs can be used for mapping genes, human identification, chimerism analysis, and many other applications.
The Human Haplotype Mapping (HapMap) Project is aimed at identifying SNP haplotypes throughout the human genome.

52. Mitochondrial DNA Polymorphisms
Sequence differences in the hypervariable regions (HV) of the mitochondrial genome

53. Mitochondrial DNA Polymorphisms
Mitochondria are maternally inherited.
There are an average of 8.5 base differences in the mitochondrial HV sequences of unrelated individuals.
All maternal relatives will have the same mitochondrial sequences.
Mitochondrial typing can be used for legal exclusion of individuals or confirmation of maternal lineage.

54. Summary
Four types of polymorphisms are used for a variety of purposes in the laboratory: RFLP, VNTR, STR, and SNP.
Polymorphisms are used for human identification and parentage testing.
Y-STR haplotypes are paternally inherited; maternal relatives have the same mitochondrial DNA alleles.
Polymorphisms are used to measure engraftment after allogeneic bone marrow transplants.

55. Summary
Single-nucleotide polymorphisms are detected by sequencing, melt curve analysis, or other methods.
SNPs can be used for the same applications as other polymorphisms.
Mitochondrial DNA typing is performed by sequencing the mitochondrial HV regions.
Mitochondrial types are maternally inherited.