DNA marker analysisMrs. StewartMedical InterventionsCentral Magnet School

Standard:Marker analysis is a technique used to determine the presence of genetic mutations associated with cancer.

Objective:Investigate the marker analysis technique and analyze results to determine the presence of a BRCA 2 mutation

BRCA 1 and BRCA 2Tumor suppressor genesRepair DNA damage and control cell growthProto-oncogenes

BRCA 280,000 nucleotides (larger than average gene)600+ mutations identified and linked to increased risk of cancerMost mutations are insertion or deletion mutations (frameshift) of more than one base

Results in a protein that is unable to help repair damaged DNA or fix mutations

Marker Analysis Technique

A technique where a gene mutation is analyzed by using a genetic marker instead of directly analyzing the gene itself.

Genetic marker: Alteration in DNA that may indicate an increased risk of developing a specific disease or disorder

STRsGenetic markers used in marker analysis are short DNA sequences called Short Tandem Repeats (STRs)STRs – region of DNA composed of a short sequence of nucleotides repeated many times.

# of repeated sequences varyAlternate forms of STRs correspond with different alleles.

STRsMost STRs occur in gene introns (non-coding regions of DNA)Does not usually affect gene functionCan use as “markers” to differentiate between different alleles for certain genes

(because genes located next to each other are inherited together.)

PCRThe region of the DNA that is the known STR marker is amplified (and the BRCA unknown gene version with it)The amplified DNA is then run on a gel.

Gel ElectrophoresisBecause different alleles have a different number of repeats present in the STR, gel electrophoresis will separate different alleles based on the number of repeats present.The more repeats present = the longer the DNA fragmentShorter DNA fragments migrate farther down the gelSo, the fragments that migrate the farthest, have the fewest STRs

Smith Family AnalysisWe will determine different alleles for each family member tested by determining the band lengths for each family member.Who was tested?

Diana –Judy’s sisterJennifer – Judy’s sisterLaura – Judy’s momJudy – Sue, Mike and Tucker’s mom

Who has the BRCA 2 mutation?Each person has 2 chromosomes

#13, so each person will have 2 alleles for the BRCA 2 gene.You will have to identify which allele is linked to the “mutant” gene by determining which alleles Jennifer and Laura have in common

Since both of them are known to carry that allele.

Analyze Judy and Diana’s results to determine if they also carry the mutated gene allele.

AnalysisDNA size markers are loaded in the first well.Use the size markers to determine the sizes of your unknown fragments.The known molecular size markers (weights in base pairs) are written beside each band.Measure the distance each unknown band migrated

DataDistance Migrated (mm) - Measure the distance in mm from the sample well to each fragment in the standard lane. Record in Table OneDistance to Reference Point - Measure the distance from the sample well to the end of the gel. Record in Table One.

This number will be the same for each size marker fragment.

Calculate the Rf of each fragment and record in Table One.

Round values to the nearest one hundredth.

DNA Size Markers

Fragment Length in

Base Pairs

Distance Migrated

(mm)A

Distance to Reference Point (mm)

B

Rf  

A ÷ B

Fragment 1 1353      

Fragment 2 1078      

Fragment 3 872      

Fragment 4 603      

Fragment 5 310      

Fragment 6 281      

Fragment 7 234      

Fragment 8 194      

Table One: