6. polymorphism

Question 1

polymorphism def

Answer 1

the occurrence in a population of two or more alleles (as long as the frequency of these variant alleles are >1% otherwise they are mutants)

Question 2

types of polymorphism

Answer 2

-protein polymorphism: eg ABO blood groups, serum proteins

-chromosomal polymorphism: regions of varying chromosomal structure (eg often on long arm of Ch9)

-DNA sequence polymorphism: variations of coding sequences

Question 3

Types of DNA sequence polymorphism (4)

Answer 3

SEQUENCE CHANGES:
1. SNP: short nucleotide P, insertion or deletion (detected via sequencing)

2. RFLP: restriction fragments length P, presence or absence of certain restrictio enzyme sites (detected via southern blot)

LENGTH CHANGES:
1. VNTR: variable nuc tandem repeats, 10-50 base repeats, (detected via southern blot or PCR)

2. STR: short tandem repeats, 1-10 base repeats (detected via PCR)

Question 4

what is used to study protein polymorphism

Answer 4

electrophoresis (or western blotting)

Question 5

why is protein polymorphism observed?

Answer 5

-if tehre is variation in coding sequence (eg base change) there might not be an effect on the protein (if the aa it codes for is the same) due to genetic code being degenerate)

however if the aa does change then a diff protein is expressed

Question 6

what is the use of DNA sequence polymorphism

Answer 6

GENETIC MARKERS: found throughout the genome, and hence if the location of the polymorphic sequence is known (ie the exact locus) it serves as a landmark or marker for locating other genes.
! each marker has diff alleles (versions)

Question 7

what is the purpose of using polymorphism as a genetic marker

Answer 7

GENERALLY:
-genome mapping
-forensic investigations
-study of evolution

CLINICALLY:
-genetic counselling
-determining susceptibility of individuals to a complex disorder
-personalised medicine (bcos determines individual reactions to a drug)

Question 8

describe RFLP

Answer 8

restriction fragments length polymorphism

-change in base can cause abolition of restriction enzyme sites along the sequence of DNA
-if you isolate a DNA and run a southern blot, exposing the DNA to restriction enzymes and a probe, the DNA will either be cut or not cut depending on if the sites are still present.
-depending on the fragments visualised you can understand if there is a changed site

Question 9

use of RFLP in labs

Answer 9

PARENTAGE TESTING:
-RFLP genotypes are inherited (one allele from each parent for each locus)
-by comparing the offspring alleles and figuring out which ones were passed by the mother, you can test for alleged paternity and see if its possible for the father to have passed the remaining allele at that locus

(this can also be used in forensic situations to match suspects to evidence found at the scene)

Question 10

describe SNP

Answer 10

single nucleotide polymorphism

-occurs roughly every 1250 bps and accounts for the 0.1% diff of the DNA of each individual
-99% of them have no biological effect
-detection by sequencing
-inherited together in blocks called HAPLOTYPES: a section of the chromosome inherited together (due to genetic linkage), in which no recombination occurs (important for mapping)

Question 11

describe STR

Answer 11

short tandem repeat (microsatellite) polymorphism

-can be repeated a diff number of times
-present throughout entire genome and if found in genes can cause disorders due to instability
-can be analysed by southern blot (but takes a long time)
-can be analysed using PCR (best method)

Question 12

describe forensic DNA testing

Answer 12

-used to match culprit from suspects from DNA evidence / link weapon, prove/disprove alibis
-sources of this DNA are from blood, semen, saliva, urine, hair follicles, teeth and bones
(a very small amount of blood can provide a sufficient DNA profile)

Question 13

why is STR good for identification?

Answer 13

number of repeats varies notably between diff individuals

Question 14

general steps in DNA analysis for identification

Answer 14

1. collection of specimen and storage
2. DNA extraction and quantitation
3. PCR amplification
4. STR analysis for interpretation of results
5. storage into database

Question 15

Describe the PCR process of STR

Answer 15

series of temperature cycles:
1. denaturing phase (separate the strands)
2. annealing with the addition of primers
3. extension of primers to create more copies
-repeating cycle to make exponential increases in the DNA strands (amplification)

!! for STR we add dye to create a fluorescent labelled product. The position of the strand where primer1 binds is the same, but the repeat regions are variable so the primer 2 region can be different. The primer positions affect the length of the PCR product.
RESULT: if the alleles are the same length = homozygote, if alleles differ = heterozygote

Question 16

how can we distinguish the sex of a DNA we are identifying

Answer 16

AMELOGENIN LOCUS (eg from blood sample)

-HUMAMEL gene (not an STR) codes for the amelogenin-like protein
-the locus is present on both X and Y Chr.
-X locus produces a PCR product of 212bps, and Y a product of 218bps.
HENCE: a female will have one peak at 212 (bcos of 2X), but a male will have a peak at 212 and another at 218 (because XY)

Question 17

what is multiplex PCR

Answer 17

PCR is used to amplify several different DNA sequences simultaneously.

Question 18

what technique follows multiplex PCR for STR identification

Answer 18

CAPILLARY ELECTROPHORESIS SYSTEM:
1. capillary filled with polymer solution (liquid agarose) - this can separate DNA based on molecular weight
2. multiplex products are denatured and put at the -ve pole
3. electrical voltage is applied and they move towards the +ve pole: smaller fragments move faster than larger ones
4. when DNA fragments pass through a detection window, a laser light excites fluorochrome (bcos these PCR products had preivously been labelled with dye)
5. RESULT: signals are captured and translated into peaks of their molecular weight (and construct allele ladder)

Question 19

What is an allele ladder

Answer 19

standards representing all alleles for each locus observed in a given population

serves as a database so that we can compare the allele peaks of diff individuals (after the capillary electrophoresis) to identify which of the allele peaks they have.
(2 peaks = heterozygotes and 1 peak = homozygote)

Question 20

STR based chimerism testing MEANING

Answer 20

-method used in cases of ALLOGENIC bone marrow transplants: a patient receives healthy HSCs from a donor and becomes a chimera (ie mixture of their own DNA and that of the donor cells).

-used to monitor the sucess of this transplant by evaluating the ratio of donor and recipient DNA in the recipient’s blood or bone marrow.

Question 21

STR based chimerism testing PROCESS

Answer 21

2 part: pretransplant and posttransplant

1. PRE: informative analysis
2. POST: engrafment analysis –> evaluation of whether all the cells of the recipient were destroyed (bcos if the old tissue still remains there is a chance of graft failure and relapse)