Genetics (General) Flashcards
Gene
- specific linear sequences of nucleotides located at a specific position (locus) along a chromosome -
- a segment of DNA that codes for a protein, which in turn codes for a trait
Allele
An alternate form of a gene that occur at the same locus of a given chromosome
Locus
a specific position on a given chromosome
Nucleotide
combination of a sugar, a phosphate group, and a base
Purine bases
Adenine (A) Guanine (G)
Pyrimidine bases:
Thymine (T) Uracil (U) in RNA Cytosine (C)
mRNA (messenger RNA)
carries the code for specific amino acid sequences from the DNA to the cytoplasm for protein synthesis. Is a single stranded linear sequence of nucleotides (replacement of thymine by uracil, which also pairs with adenine)
tRNA (transfer RNA)
carries the amino acid groups to the ribosome for protein synthesis
rRNA (ribosomal RNA)
exists within the ribosomes and is thought to assist in protein synthesis
Transcription
RNA is made from DNA
Translation
Proteins are made from the message on the RNA
RNA Polymerase
is the enzyme responsible for DNA Transcription
redundancy
Amino acids can be specified by several different codons called
Splicing
process where introns are cut out (process that takes place during mRNA processing)
Intron:
are the regions that are not represented in the mRNA
Exon:
regions that are represented in mRNA and specify protein-coding sequences and the sequences of the untranslated 5’ and 3’ regions
Probe:
- A small slice of DNA whose sequence is complementary to the DNA sequence under study
- Probes may be natural or artificially manufactured
Vector:
an independent, self-replicating element
DNA Library:
-The total amount of bacterial clones harboring recombinant vectors is termed DNA library
Plasmid (vectors)
- Are round molecules of double-stranded DNA that are found naturally in bacteria
- Are used to move genes from cell to cell
How is DNA purity assessed
by the ratio of its optical density at 260nm to that at 280nm, with the OD 260/280 ratio for pure DNA being 1.8
Low ratios of DNA purity
<1.6 DNA is contaminated with protein or materials used in the isolation procedure
High ratios of DNA purity
>2.0, indicated that the DNA is contaminated with RNA
PCR steps
1) Denature 2) Annealing 3) Extension
Reverse Transcriptase (RT) - PCR
When RNA is reverse transcribed, the resulting DNA is a single-stranded complementary copy of the RNA and is referred to as complementary DNA (cDNA)
Gel Detection
Detects bands by staining with ethidium bromide, during or after electrophoresis Amplified DNA analyzed by agarose gel electrophoresis in presence of ethidium bromide which binds to double stranded DNA and is visible under UV light
Real-Time PCR
employs one or more “probes” that fluoresce only when the correct amplicon is present.
RFLP
restriction fragment length polymorphisms
VNTR
variable number tandem repeats (8 - more than 50 base pairs)
STR
short tandem repeats (1- 8 base pairs)
SNP
single nucleotide polymorphisms
Nucleic Acid Amplification Testing (NAT)
- Transcription-mediated amplification
- This testing allows specific sequences to be multiplied rapidly and precisely
- Before amplification, viral RNA must be converted to DNA
Restriction Fragment Length Polymorphisms (RFLP)
- sizes are altered by changes in or between enzyme recognition sites.
- RFLP genotypes are inherited -for each locus, one allele is inherited from each parent
RFLP uses
- gene mapping
- analysis linkage
- analysis characterization of HLA genes in transplantation (tissue typing)
- paternity testing
- forensic science
Polymorphism
- a sequence difference in DNA compared to a reference standard that is present in at least 1-2% of a population
- can be single bases or thousands of bases
- may or may not have phenotypic effects
New polymorphisms are created by: (7)
- Missense mutations
- Nonsense mutations
- Mutations resulting in alternate RNA splicing
- Exon insertion
- Gene deletion
- Chromosome translocation
- Gene recombination
3 possible outcomes can follow the SUBSTITUTION of a single nucleotide:
- Silent
- Missense
- Nonsense
Silent Mutation
- substitution causing no effect on protein being synthesized, because codon would translate to the same amino acid
- occurs when one base is substituted for another, but the effect does not change the codon translation
Missense Mutation
- Substitution of an amino acid that changes the nucleotide configuration and the protein being synthesized
- occurs when a substitution could result in a sequence that changes the product of the codon
Nonsense Mutation
- A substitution which results in producing a stop codons.
- This means that the protein synthesis is truncated, and does not produce the correct protein
- No protein production occurs beyond this point.
How are STR alleles analyzed?
- by fragment size (Southern blot)
- by amplicon size (PCR)
SNPs can be used for:
mapping genes human identification chimerism analysis
SNPs are detected by:
sequencing melt curve analysis
Why genotyping of red cell antigens may be more efficient than traditional serologic typing
-When a patient has received multiple RBCs and it is not possible to distinguish the patient’s own red cells from transfused red cells -Occasionally, the genotype may not correlate with the phenotype. -Focuses on known polymorphisms but does not provide the entire sequence of the gene or its regulatory regions.
The most common nucleotide change leading to the expression of a blood group antigen
Single Nucleotide Polymorphisms
Minisatellites
variable number of tandem repeat (VNTR) loci consist of tandem repeats of a 6-100 base pair sequence
Microsatellites
short tandem repeat (STR) loci consist of tandem repeats of short (4 base pair) sequence
DNA fingerprint
So much variation between individuals that chances are very low that same number of repeats will be shared by two individuals
Restriction Endonucleases
- protect bacteria from viral infection by degrading viral DNA after it enters cytoplasm
- recognizes only a single specific nucleotide sequence
- enzyme cleaves DNA strand wherever recognized sequence occurs, generating # of DNA fragments whose length depends upon # and location of cleavage sites
Common Restriction Endonucleases
- Eco RI (E. coli)
- Hind III (H. influenzae)
- Hpa I (H. parainfluenzae)
Restriction enzymes cut double stranded DNA at specific sites in one of three ways.
DNA Cloning
- DNA containing gene of interest inserted into vector which is self replicating genetic element such as a virus (eg, bacteriophage lambda) or a plasmid (small circular molecules of double stranded DNA that occur naturally in bacteria)
- After gene inserted into DNA of the vector, recombinant DNA can be introduced into bacterial host where it undergoes replication
- Permits genetic engineering, including production of recombinant proteins and gene therapy
Low Resolution Genotyping Methods
- Gel-based methods
- SSP-PCR for known SNPs
- PCR-RFLP for known SNPs
- High melt resolution analysis
Medium Resolution Genotyping Methods
- Arrays
- Immucor BeadChip and Grifols IDCore
- Taqman genotyping
- Single base extesion (SBE) using MALDI-TOF
High Resolution Genotyping Methods
- DNA sequence analysis
- Exon scanning
- cDNA analysis
- Next Generation Sequencing
Method Type (AABB Molecular Testing Standard)
- Novel test method
- At least 20 samples
- At least a wild-type and heterozygous sample
- New test, existing test method (in the lab)
- At least a wild-type and heterozygous sample
DNA Microarrays
- Also called gene chips
- DNA is generated by PCR and the nucleotide sequences are then probed
- Method involves tens of thousands of separate DNA molecules spotted or synthesized on small area of solid support (plated into wells on nonpermeable support such as glass
- The result is a diagram of a gene expression profile
- Used for comparative genomics and genotyping (applied to blood groups)
- Determine genotype and/or phenotype of red cell antigens
Gene Therapy
- refers to the introduction of nonself genetic material into cells to treat or prevent disease
- Can be used to replace defective gene, leading to increased production of a specific protein
Gene Rearrangement
- Gene rearrangements are normal events that occur in lymphocytes
- Antibody genes [immunoglobulin heavy chain genes, immunoglobulin light chain genes (κ, λ)] and T cell receptor genes (α, β, γ, δ) rearrange
- Rearrangement occurs independently in each cell
Northern Blot
- Used to study gene expression
- RNA is analyzed
- Technique uses electrophoresis and detection with a hybridization probe (can be made from DNA or RNA)
Southern Blot
- Used to check for the presence of a DNA sequence in a DNA sample (RFLP analysis)
- Combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization
- Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments
Western Blot
- Used for detecting specific proteins in a given sample of tissue or cells
- The proteins of the sample are separated using gel electrophoresis (SDS-PAGE)
Target and Probe of Southern blot
- Target: DNA
- Probe: Nucleic acid
Target and Probe of Northern blot
- Target: RNA
- Probe: Nucleic acid
Target and Probe of Western blot
- Target: Protein
- Probe: Protein
Target and Probe of Southwestern Blot
- Target: Protein
- Probe: DNA
Molecular Events that give rise to Blood Group Antigens
(8)
- Deletions – gene, exon or nucleotide
- Duplication events
- Insertions – nucleotide(s)
- Gene Conversion (template)
- Changes to Regulator/Modifying Genes
- **SNPs are by far the most common nucleotide change leading to the expression of a blood group antigen***
- Errors in DNA replication can generate new blood group antigens
- Nucleotide changes are inherited and predict the expression of an antigen
Challenges of Serologic Typing
- Serologic reagents vary or are unavailable: Monoclonal, Polyclonal, blends or patient source
- Antigen variants can be missed: ex. U variants
- Expression level can hamper detection (ex. Weak D)
- Cross-reactivity: ceHAR, ceCF
Genetic information can be used for diagnostic purposes:
- Fetal genotyping
- Multi-transfused recipient genotyping
- Blood donor extended blood group genotype screening
Indications for genomic RBC typing
- Discrepant serologic results
- Cannot get red cell sample for RBC phenotype
- When zygosity matters
- No antisera exists
- Post-transfusion sample
- Interfering antibodies
- Serologic typing unable to detect
- Desire to RBC type large numbers of people
