Molecular Genetics Lecture 4 - DNA Analysis Techniques

What are pathogenic variants?

disease causing mutations in humans

Who is able to diagnose diseases?

geneticists, physicians, genetic counselors

What causes 70% of human diseases?

single nucleotide polymorphisms (SNPs)

What causes 20% of human diseases?

insertion and deletions (INDELs)

What are the 3 types of large DNA sequence changes?

copy number variants (CNVs), large insertions, trinucleotide repeats

What is a copy number variant (CNV)?

>1000 bp INDELs

what are large insertions?

transposons

What is an example of a trinucleotide repeat?

CAG causes Huntington disease

What are the components required for PCR?

template DNA, buffer, short oligonucleotide primers, deoxyribonucleoside triphosphates, thermostable DNA polymerase Taq, PCR machine/Thermocycler

4 steps of PCR

denaturation, annealing, extension, cycling

what happens during denaturation?

heating the mixture to 92-95 degrees Celsius separates the two strands of the DNA

what happens during annealing?

cooling the mixture (45-65 degrees Celsius) allows the primers to bind or anneal to complementary sections of single-stranded target DNA

what happens during extension?

heating the mixture to 72 degrees Celsius causes the Taq polymerase to synthesize the complementary DNA strand from the dNTPs, starting at the primer

what happens during cycling?

repeat the above to make more copies

__ cycles increases DNA copies by ~10⁶

20

what are some applications of PCR?

making copies of DNA sequences (cloning), detect and quantify gene expression (mRNA), DNA sequencing, detection of DNA methylation

What is Southern blot analysis used for?

used in estimating copy numbers of a gene, genotyping using restriction fragment length polymorphism (RFLP) and DNA fingerprinting

4 steps of Southern blot analysis

1. DNA is digested with restriction enzymes to fragment the genomic DNA.

2. DNA molecules can be separated by size by gel electrophoresis using agarose gel.

3. DNA molecules can then be transferred from the gel onto a nitrocellulose or nylon membrane using a technique called a Southern blot.

4. DNA on the membrane can be denatured and hybridized with DNA probe radioactively labeled with 32Phosphorus

How can Southern Blot be used to diagnose Huntington Disease (HD)?

HD is a polyglutamine disorder. CAG codon codes for the amino acid glutamine. Increased number of CAG repeats in the first exon of HT gene leads to increased number of glutamine amino acids in the protein

What does CAG repeats mean?

copy number and onset of HD disease

HD diseases shows reduced ______ and is a _____ ____ disorder

reduced penetrance, late onset disorder

what do penetrance and disease onset depend on?

CAG repeat copy number

What is used to detect chromosomal changes (insertion, deletion, duplication, etc)?

DNA FISH

What is Fluorescence In situ Hybridization?

FISH

what are the steps for FISH?

1. cells from mitosis or interphase are fixed to preserve the integrity on a slide.

2. denature the chromosomal DNA to make it single stranded.

3. single stranded DNA is used as a “probe.”

4. probe is fluorescently labeled.

5. when a probe binds to a target DNA by base complementarity, it glows

what is chromosome painting?

multiple probes labeled with different colors to distinguish regions

what can be detected using FISH?

trisomy 21 because it is a chromosomal abnormality

____ is used to detect large detections, not targeted

FISH

____ is used for small detections, not targeted

CGH

what does CGH stand for?

comparative genomic hybridization

what are CGH arrays?

DNA microarray, whole genome tilting array, genome-wide detection of large insertions/deletions

__ is very small and targeted and can only detect known diseases

DNA SNP microarray

what is DNA SNP microarray?

genome-wide detection of disease causing mutations/alleles of specific genes such as BRCA-1, sickle cell, cystic fibrosis