BITC2441 Molecular Biology Techniques
Name __________________
Molecular Diagnostics by Buckingham & Flaws
Guided Lecture Notes
Chapter 11. Human Polymorphisms and Human Identification
After reading the whole chapter, answer the following questions into your lecture notebook. Leave spaces for additional
information that might come up during lecture. This preview of the lecture and will allow you to minimize the time needed to
enter lecture notes into your notebook during the lecture. With your notes outlined ahead of time, you will be able to ask
questions during lecture over information that you need help with.
After answering all the questions, sign the question sheet and circle the questions that you want to have emphasized during
lecture, either because you are unsure of your answer or because you need clarification. Hand in this sheet with your circled
questions at the beginning of class. You will not turn in your lecture notebook; you will be given a completion grade for your
work.
TYPES OF POLYMORPHISMS
1.
Compare the definition of a polymorphism and a mutation.
2.
On the average, how frequently in a nucleotide sequence do SNPs occur in the human genome?
a. More SNPs occur in noncoding regions than in coding regions of the genome. What type of genes are notable
exceptions to this rule?
b. What roles do HLAs play in the immune system and tissue transplants?
3. The human genome is 98% noncoding sequences and much of the noncoding regions harbor block of repetitive
elements. These repeated elements can be tandem repeats or interspersed repeats. Interspersed repeats are often
referred to as mobile elements or transposable elements because they can be copied and spread (“jump”) to random
locations in the genome. How do LINEs and SINEs differ with respect to:
a. length
b. gene content
c. reverse transcriptase
d. number of repeats in the human genome
4.
What are some differences between STRs and VNTRs?
RFLP TYPING
5.
What are some types of sequences differences that can give rise to RFLPs?
6.
Describe the steps in preparing a RFLP.
a. What enzymes and detection labels are used in this process?
b. What type of sequence is the probe designed to complement?
c. Explain what a single locus probe is, compared to a multiple locus probe, and how they affect the number of bands
that show up in a RRLP fingerprin.
d. How can RFLP analysis be used to establish parentage?
e. How many RFLP loci have been developed for DNA typing in humans?
f. Who provides reference standards for RFLP DNA fingerprinting?
7.
Explain how RFLPs have been used as genetic markers for mapping genes.
8.
Explain why it is easier to exclude than to include human identity using DNA fingerprinting.
9.
What are some serious limitations and drawbacks to the use of RFLPs for identity testing?
STR TYPING BY PCR
10. How does minisatellite DNA differ from microsatellite DNA?
a. How do microvariants repeat units differ from those normally found in STRs?
b. Why are STRs amplified better by PCR than are VNTRs?
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11. What are some advantages of PCR profiling of DNA, compared to RFLP analysis?
a. What is an allelic ladder, and why is one required (along with an internal size standard) for STR analysis?
b. How many different alleles can a given STR have in a population? In an individual?
c. How can multiple STR loci be run in a single PCR reaction? How can overlapping STR loci be discerned?
12. What is the theoretical lower limit to the number of cells required for identity testing by STRs?
a. What amount of DNA does this lower limit amount to in human cells?
13. Who has developed a uniform nomenclature of STR loci?
a. Who is HUGO, and what is their mission? Unfortunately, any given gene can have multiple names and symbols in
use, due to multiple conventions in use by different groups who have discovered the genes. In an effort at
standardization, The HUGO Nomenclature Committee has a sponsored website cross-referencing gene symbols
and names for almost 24,000 human genes (www.gene.ucl.ac.uk/nomenclature/). The standardized symbol for a
human gene uses italicized capital letters.
b. What is the recommended convention for naming STR loci with respect to:
Gene locus
Intron location
Non-gene locus
c.
d.
Where are newly-discovered STR names registered?
Where are commonly-used STR loci archived in an online database?
14. The amelogenin locus has only two alleles. Why was this locus included in the FBI Combined DNA Indexing System
(CODIS)?
15. Who supplies Standard Reference Material (SRM) for identity and paternity testing laboratories?
16. How have databases for STR allele frequencies been developed for identity test analyses?
a. How is a STR locus genotype determined and named?
b. How is genetic concordance established for a STR profile?
17. What are some advantages of running internal standards in a STR analysis?
a. How is a “bin” created for STR alleles on a given capillary gel electrophoresis instrument?
b. What are some artifacts of gel electrophoresis that can give rise to an uncertainty window associated with STR
allele migration rates?
c. What types of enzyme anomalies can give rise to a background of alternative alleles on a STR gel?
d. What types of STRs have the greater problem of stutter?
18. Explain how degraded, compromised, and mixed samples can interfere with positive identification by match probability.
a. How can a lab insure that its criteria for interpretation of STR profiling results are valid?
b. How can external proficiency testing be done?
19. In matching STR profiles, how is a likelihood ration computed and interpreted?
a. How is a paternity index computed and interpreted?
b. How is a “probability of paternity” different from a CPI?
c. Why is a sibling test more difficult to analyze than is a paternity test?
d. How can kinship indexes be improved?
Y-STR
20. What is unique about the Y chromosome haplotype?
a. How does the inheritance pattern of the Y-STR DNA give rise to special applications?
b. How does the mutation rate for the Y chromosome compare to that of the autosomes, and what application does
this give rise to?
21. How is haplotype diversity (HD) determined for a given set of Y-STR loci?
a. How can the discriminatory capacity (DC) be improved for a given haplotype?
b. Why is the product rule of probability estimations inappropriate in Y-STR haplotype frequencies?
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ENGRAFTMENT TESTING USING DNA POLYMORPHISMS
22. What is the major advantage of an autologous, rather than an allogeneic, transplant donor, and why is autologous
donation unusual?
a. What tissue is primarily used for transplantation of hematopoietic stem cells and what sorts of disorders call for this
type of treatment?
b. Post-transplant risk of hematopoietic stem cell graft failure and mortality are affected by quantitative and qualitative
characteristics of donor-recipient HLA allele mismatching. What cellular processes occur during engraftment, and
what are some strategies to avoid graft-versus-host-disease (GVHD)?
c. Only about 30% of patients requiring a bone marrow transplant have a sibling with HLA alleles matching their own
sufficiently. Registries of volunteer bone marrow donors and cord blood exist (www.marrow.org). Currently, nearly
65% of patients can find an acceptable donor among more than 4 million individuals registered in the US-based
national marrow donor program. How can genetic analysis help to screen potential tissue donors for immunological
compatibility?
d. How can molecular methods be used to monitor the engraftment period? How is analysis of informative alleles
different in this case from how STRs are analyzed for identity testing?
e. Why is it best to locate informative loci in recipients prior to transplantation?
LINKAGE ANALYSIS
23. What are 3 sources of information that allows mapping of genes to STRs?
a. Why is the linkage of a genetic disorder to a STR of practical value?
b. Why are STR linkage studies of unrelated individuals in populations more difficult than tracing linkage in a
pedigree?
SNPs
24. About how frequently in nucleotide sequences of the human genome are sequence differences encountered?
a. What type of sequence difference is the majority of these?
b. About how many SNPs have there been estimated that each individual carry in their genome?
c. How many SNPs have been identified so far?
d. What proportion of SNPs have a biological effect?
25. What causes linkage disequilibrium between SNPs?
a. What is the approximate length of a SNP haplotype?
b. What is a haplotype map (HapMap) and of what practical use is one in the medical field?
c. What types of technology are best suited to detect SNPs?
MITOCHONDRIAL DNA POLYMORPHISMS
26. Describe the size and structure of the mitochondrial genome.
a. What asymmetry gives rise to a H- and a L-strand?
b. The mitochondrial genome is double-stranded for most of its length except for the control region (D loop) where
replication and transcription control sequences are found. Genes are transcribed into polycistronic RNAs. What
types of genes are transcribed and how many promoters are there for these genes?
c. Each mitochondrion has multiple copies of chromosomes, and each cell contains a variable number of mitochondria
depending on the energy requirements of the cell. Some cell types may contain up to several thousand copies of
mitochondrial DNA. What sorts of testing applications is this abundance of copy number convenient for?
d. Mitochondria are maternally inherited. What sorts of testing applications is this inheritance pattern convenient for?
e. Which part of the mitochondrial genome carries the most polymorphisms, and how many differences in nucleotide
sequences generally occur between unrelated individuals?
27. What is heteroplasmy and how does this phenomenon interfere with the use of mtDNA sequencing for identity testing?
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KEYWORDS
SNPs
human leukocyte antigen (HLA)
long interspersed nuclear elements
(LINEs)
short interspersed nuclear elements
(SINEs)
mobile / transposable elements
reverse transcriptase
microsatellites/minisatellites
variable number of tandem repeats
(VNTRs)
short tandem repeats (STRs)
microvariants
loci
restriction fragment length
polymorphisms (RFLPs)
one-locus/multilocus probes
allelic ladder
Human Genome Organization
(HUGO)
Combined DNA Indexing System
(CODIS)
National Institute of Standards and
Technology (NIST)
Standard Reference Material (SRM)
STR locus genotype
STR genotype/ STR profile
concordance/inclusion/exclusion
bins/binning
dye blobs
stutter
fixed/floating bins
locus-specific brackets
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Hardy-Weinberg equilibrium/ Hardy
Weinberg Law
product rule of probability
linkage equilibrium
likelihood ratio
combined paternity index (CPI)
probability of paternity
full/half sibling test
kinship/sibling/combined sibling index
avuncular test
Y-STRs
haplotype diversity (HD)
discriminatory capacity (DC)
paternity index (PI)
autologous/allogeneic donor
myeloblative conditioning
engraftment
graft-versus-host-disease (GVHD)
human leukocyte antigen (HLA)
genetic chimera
donor-/recipient-informative loci
single nucleotide polymorphisms
(SNPs)
linkage disequilibrium
tag SNPs
ancestral haplotype
haplotype map (HapMap)
heavy-/H-strand
light-/L-strand
control region
displacement-/D-loop
hypervariable regions I & II
(HVI & HVII)