Molecular Genetics Methods Flashcards
Basics Principles:
Method for Cleavage of the DNA
Restriction endonucleases
Basics Principles:
Method for Amplification
PCR
Basics Principles:
Method for Separation and visualization
Electrophoresis
Basics Principles:
Method for Visualization by hybridization (using probe)
Blot (southern) / Microarray / FISH
Basics Principles:
Method for Sequencing
Sanger / NGS
Detection of SNV: Allele specific hybridization and amplification - Which methods are used here?
Allele specific hybridization: Southern blot, microarray Allele specific amplification: PCR
Detection of SNV: What is PCR-RFLP?
3 steps:
1) PCR amplification of DNA sequence of interest.
2) Cleavage of PCR product by allele specific restriction endonuclease.
3) Gel electrophoresis to identify of cleavage pattern (genotype).
What is Southern Blot?
A procedure for identifying specific sequences of DNA, in which fragments separated on a gel are
transferred directly to a second medium on which assay by hybridization (i.e., by a probe) may be carried
out.
What is Southern Blot used for? examples
Southern Blot is used for:
− Genetic testing, e.g., in homologous alleles
− Restriction fragment length polymorphisms (RFLPs)
− Variable number of tandem repeats (VNTR)
What is ASO Test?
Allele-Specific Oligonucleotide (ASO) Test:
ASO probes have the ability to reliably detect single nucleotide changes, unlike Southern Blot which is
unable to distinguish small changes in the DNA.
What is ASO Test used for? example
Cystic Fibrosis as ASO test usage:
-Oligonucleotide probe (ASO) is complementary to the exact DNA sequence of the allele with the 508
deletion.
-DNA is obtained from all family members and spotted onto a Southern hybridization filter.
-Filter is probed with a radioactive ASO.
-Hybridization of the ASO to the filter is indicated by a spot on the autoradiogram, which indicates the
presence of the 508 allele.
What can ASO be combined with to detect 508 deletion in carriers Cystic Fibrosis?
Pedigree analysis of Proband’s Family
What is DNA Microarray ?
DNA microarray (gene chip, DNA chip, or biochip): either measures DNA or uses DNA as a part of its detection system. In each of the spots in this array, a known DNA sequence or gene is orderly arranged.
What are the types of DNA microarray?
types of DNA microarray:
- cDNA microarrays- uses complementary DNA strands formed by transcription of mRNA
- SNP microarrays- used to detect variants within a population
What are examples of Polymerase Chain Reaction types?
3
examples of Polymerase Chain Reaction types:
- RT-PCR
- qPCR
- MLPA
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR):
Used for? How (Generally)?
RT-PCR is used for the analysis of mRNA expression. by reverse transcription of mRNA (less stable to analyze) a cDNA (copy DNA) can be analyzed.
Real-Time / Quantitative PCR (qPCR):
Used for? How (Generally)?
• Used to detect the PCR product as it is produced (as the fluorescent signal is generated), thus follow the
reaction in real-time.
• This allows accurate quantification of the amplified DNA by means of fluorescence measurements.
• Enables both quantitative analysis of DNA,
as well as the determination of the amount of RNA.
Real-Time / Quantitative PCR (qPCR):
Applications?
Real-Time / Quantitative PCR (qPCR):
- quantitative analysis of DNA: bacterial DNA, human gene amplification (SNPs)…
- determination of the amount of RNA: measurement of gene expression, viral RNA (qRT-PCR fro COVID-19)
Multiplex Ligation-dependent Probe Amplification (MLPA):
Used for? How (Generally)?
Amplification of multiple target DNA sequences using only a single primer pair.
The MLPA probes that hybridize to the target sequence are amplified and not the target sequences.
Analyzed by capillary electrophoresis - comparing the peak pattern obtained to that of reference samples
indicates which sequences show aberrant copy numbers.
Multiplex Ligation-dependent Probe Amplification (MLPA): Applications? Examples
- Duchenne muscular dystrophy (DMD)
- Hereditary breast/ ovarian cancer (BRCA)
- Hereditary colorectal cancer syndromes (APP)
- SNP- detection of a CHEK2 variant (1100delC)
Sanger Sequencing:
Aim?
looking for sequence differences in a not too large, known gene (and not looking for a specific known
mutation or polymorphism). (~1000Bp)
Sanger Sequencing:
What is detectable?
Detectable: point mutations, small insertions, deletions, duplications, heterozygosity, mitochondrial
heteroplasmy.
Next Generation Sequencing (NGS):
Aim?
Also known as ‘high-throughput sequencing’, parallel sequencing of millions of DNA. 1 run- hundreds Mb or few Gb sequence.Nowadays used more or less for research, for whole genome sequencing, in the future will be used for diagnostics as well.
Whole Exome Sequencing (WES) advantages compared to Whole Genome Sequencing (WGS):
- Identifies variants across a ____ range of clinical applications
Whole Exome Sequencing (WES) advantages compared to Whole Genome Sequencing (WGS):
- Identifies variants across a WIDE range of clinical applications
Whole Exome Sequencing (WES) advantages compared to Whole Genome Sequencing (WGS):
-Achieves comprehensive coverage of _____ regions
Whole Exome Sequencing (WES) advantages compared to Whole Genome Sequencing (WGS):
-Achieves comprehensive coverage of CODING regions
Whole Exome Sequencing (WES) advantages compared to Whole Genome Sequencing (WGS):
-Provides a ___-effective alternative to whole-genome sequencing (4–5 Gb of sequencing per exome compared to ~90 Gb per whole human genome)
Whole Exome Sequencing (WES) advantages compared to Whole Genome Sequencing (WGS):
-Provides a COST-effective alternative to whole-genome sequencing (4–5 Gb of sequencing per exome compared to ~90 Gb per whole human genome)
Whole Exome Sequencing (WES) advantages compared to Whole Genome Sequencing (WGS):
-Produces a _____ , more manageable data set for _______analysis compared to whole genome
approaches
Whole Exome Sequencing (WES) advantages compared to Whole Genome Sequencing (WGS):
-Produces a SMALLER , more manageable data set for FASTER analysis compared to whole genome
approaches
MammaPrint (Macroarray):
Use? Range of genes detected?
MammaPrint (Macroarray):
Only used to analyze early-stage breast cancers; can detect 70 genes.
General Terminology:
Probe
Probe:
a cloned DNA or RNA molecule, labeled with radioactivity or another detectable tracer, used to
identify its complementary sequences by molecular hybridization; also, the act of using such a molecule.
General Terminology:
Hybridization
Hybridization:
the act of two complementary single-stranded nucleic acid molecules forming bonds
according to the rules of base pairing (A with T or U, G with C) and becoming a double-stranded molecule.
Usage: “The probe hybridized to a gene sequence.”
General Terminology:
Primers (for PCR)
Primers (for PCR):
two oligonucleotides, one on each side of a target sequence, designed so that one of the
primers is complementary to a segment of DNA on one strand of a double-stranded DNA molecule and the
other is complementary to a segment of DNA on the other strand. A specific pair of primers serves to prime
synthesis of DNA in a PCR reaction.
Usage: “I designed primers for PCR.”
General Terminology:
Oligonucleotide
Oligonucleotide:
a short strand of nucleic acid, ranging in length from a few base pairs to a few dozen base
pairs, often synthesized chemically; often referred to as an oligo or oligomer. The number of bases is often
written with the “-mer” as a suffix: a 20-mer.
General Terminology:
Genetic marker
Genetic marker:
a sequence variation with a known location on a chromosome that can be used to identify
individuals, with a relative high chance to differentiate between different alleles on homologous
chromosomes. Those are microsatellites (STR) and SNPs/SNVs.
General Terminology:
Restriction endonucleases
Restriction endonucleases:
enzymes that recognize specific double-stranded DNA
sequences (site) and cleave the DNA at or near the recognition site.Usually several cuts are done since the recognition site is found at couple of places on the DNA.Usage: “a restriction enzyme digest” (or just “a restriction digest”) or “the restriction enzyme EcoRI.”
General Terminology: Complementary DNA (cDNA)
Complementary DNA (cDNA):a synthetic DNA made by reverse transcriptase, a special DNA polymerase enzyme that uses messenger RNA (mRNA) as a template; used to refer to either a single-stranded copy or its double-stranded derivative. Usage: “a cDNA clone,” “a cDNA library,” or “to isolate a cDNA.”
General Terminology:
Clone
Clone:
a recombinant DNA molecule containing a gene or other DNA sequence of interest; also, the act of
generating such a molecule.
Usage: “to isolate a clone” or “to clone a gene.”
General Terminology:
Host
Host:
the organism used to isolate and propagate a recombinant DNA molecule, usually a strain of the
bacterium Escherichia coli or the yeast Saccharomyces cerevisiae.
Usage: “What host did they clone the cDNA in?”
General Terminology:
Insert
Insert:
a fragment of foreign DNA cloned into a particular vector.
Usage: “They purified the insert.”
General Terminology:
Vector
Vector:
the DNA molecule into which the gene or other DNA fragment of interest is cloned; the resulting
recombinant molecule is capable of replicating in a particular host. Examples include plasmids,
bacteriophage lambda, and bacterial artificial chromosomes (BACs).Usage: “a cloning vector.”
Capillary electrophoresis
separate mixtures of DNA, RNA, or proteins based on molecular size and charge. In contrast to conventional gel electrophoresis, which separates molecules as they migrate through a slab gel matrix, capillary electrophoresis separates molecules as they travel along the inside of a small capillary tube that is filled with a conductive, liquid buffer, rather than a gel.
Northern blot: What is filtered?
RNA
Western blot: What is filtered?
Protein
Southern blot: What is filtered?
DNA
