Week 6 Flashcards
Descriptions of the Abdomen
Flat
Scaphoid
Rounded
Protuberant
9 regions of Abdomen
Upper
1) R Hypochondriac
2) Epigastric
3) L Hypogastric
Middle
4) R Lumbar
5) Umbilical
6) L Lumbar
Lower
7) R Iliac/Inguinal
8) Hypogastic/Suprapubic
9) L Ilian/Inguinal
4 Quadrants of Abdomen
RUQ
RLQ
LUQ
LLQ
Organs located in R Hypochondriac Region:
Liver
Gallbladder
R Kidney
Hepatic Flexure of LI
Organs located in L Hypochondriac Region:
Stomach
Liver (tip)
Spleen
Pancreas (tail of)
L Kidney
Splenic Flexure of LI
Organs located in Epigastric region:
Abdominal esophagus
Stomach
Pancreas
SI
Liver
Galbladder
R/L Kidneys
R/L Adrenal Glands
Organs located in R Lumbar region:
Liver
Gallbladder
R Kidney
Ascending colon
SI
Organs located in Umbilical region:
Stomach
Pancreas
Transverse colon
SI
R/L Kidneys
Organs located in L Lumbar region:
L Kidney
Descending Colon
SI
Organs located in R Iliac/Inguinal region:
SI
Appendix
Cecum
Ascending Colon
R ovary and uterine tube
Organs located in Hypogastric/Suprapubic:
SI
Sigmoid Colon
Rectum
Bladder
Uterus/Ovaries/Uterine tubes
Prostate/Seminal Vesicles/Vas deferens
Organs located in L Iliac/Inguinal
Descending colon
Sigmoid colon
SI
L ovary/uterine tube
Normal Liver span at midsternal line
4-8 cm in midsternal line
Normal Liver span at midclavicular line
6-12 cm in R midclavicular line
Visceral peritoneum:
Peritoneum surrounding visceral organs within the abdominal cavities
Parietal peritoneum:
Peritoneum lining the walls of the abdominal cavity
Uses for DNA Technology
1) Dx genetic conditions
2) Predict multifactoral diseases
3) Preventative med
4) Predict drug responses
5) Manufacture of biopharms
6) Cancer genomics - prognosis, treatments
7) Genomic editing/Gene therapy
Techniques to see DNA/RNA
Light Microscopy
Electrophoresis
Blotting
DNA sequencing
Ways to see DNA with “naked” eye
Compact DNA with dyes/proteins
Cut and dye DNA
Copy DNA with dye attached
DNA/RNA and Light Microscopy
Chromosomes locked in metaphase
Able to visualize histones
DNA partially denatured then labelled with probe
DNA/RNA and Sequencing
DNA extended with DNA polymerase
Terminated with 2’,3’ dideoxynucleotide
DNA/RNA and Electrophoresis
Separation of DNA/RNA by size
DNA/RNA and Blotting
Immobile DNA/RNA detected via specific sequence
Hybridizes with Probe that is tagged and can be visualized
Purpose of Light Microscopy and DNA?
Looks for LARGE abnormalities
Trisomy/Monosomy, Ring structures
Describe non-denaturing gels:
Detects larger differences or secondary structure effects
Ranges from 50,000 bp - 100 bp
Purpose of Electrophoresis and DNA?
Purpose of Blotting and DNA?
Purpose of Sequencing and DNA?
FISH is used to detect?
Chromosomal rearrangements
Large insertions/deletions in chromosomes
Trisomy/monosomy
Fluorescent In Situ Hybridization
FISH
Multiple probes added to detect multiple sequences
Types of Gel for Electrophoresis
Denaturing
Non-denaturing
Describe denaturing gels:
Detection of single nucleotide differences
Ranges from 1-2000 nucleotides
Denaturing gels for electrophoresis are useful for?
Sequencing
Copy number variation
Other analysis for differentiation in length of NA
DNA size determined via
Electrophoresis
Huntingtons Disease caused by
CAG repeats within the htt gene
Techniques for proteins
Microscopy
Blotting
Sequencing
Purpose of Sequencing and Proteins?
Chemical removal of one AA at a time
Determine AA
Edman Degradation and Mass spec.
Purpose of Blotting and Proteins?
Western: separate proteins and probe with another protein
ELISA: one protein in spot; attached to matrix; 2nd protein binds to first; probe binds to 2nd protein
Purpose of Microscopy and Proteins?
Look at stained proteins
Gram stained or Hematoxylin and Eosin
DNA Blotting Technique
Cleaved by size
Fragments transferred to matrix
2nd DNA sample labeled with dye/radioactivity
Hybridize/bind probe to first DNA
Analysis
Allele Specific Oligonucleotide Probes
- Synthesized chemically
- Up to 100 bp
- Hybridize to cDNA
- Differentiates single base pair with probe
Allele Specific Oligonucleotides used for?
Primers
ID alleles on blot
Types of Blotting Techniques:
Southern
Northern
Western
Dot
Microarray
Use of Southern Blot
Electrophoresis of DNA
Separate by size
Labelled with DNA/RNA
Use of Western Blot
Electrophoresis of protein
Separate by size
Labelled with antibody (Ab)
Use of Northern Blot
Electrophoresis of RNA
Separate by size
Labelled with DNA/RNA
Use of Dot blot
DNA/RNA used without size separation
Probe added with labelled DNA/RNA
Use of microarray
Visualization of macromolecules
(RNA, DNA, carbs, proteins, lipids, tissues, viruses)
Multiple tiny dots at one time
Southern blotting used to detect?
Restriction Fragment Length Polymorphisms (RFLP)
Dot blots used for?
Tell relative amount of DNA/RNA with specific sequence
Small as single nucleotide polymorphism
Northern blotting used for?
Detect lengths of RNA
Sanger DNA Sequencing Method
Single strand DNA of unknown sequence used as template
Primers added with labelled deoxynuclotides as well
Split sample into 4 tubes
Add one of 4 dideoxynuclotides to each tube
Allow synthesis to continue until it stops
Read electrophoresis gel
Why do dideoxynucleotides stop DNA synthesis?
No 3’ hydroxyl group to bind to to keep adding nucleotides to chain
Uses of Sequencing in terms of Discovery tool:
1) Single gene disorders
2) Polymorphisms
3) Protein prediction
4) Variants in polygenic disorders
5) Localize genes from linkage
Uses of Sequencing in terms of Diagnostic tool:
1) Genetic testing
2) Genotyping
3) IVF
4) ID Infectious pathogens
Uses of Sequencing in terms of Predictive tool:
1) Susceptibility
2) Drug sensitivity/insensitivity
3) Treatment response
4) Morbidity
Best ways to amplify DNA
PCR
Recombinant DNA cloning
cDNA
ASO
Define: Recombinant DNA
DNA inserted into vector from another organism
Is grown in organism that can replicate vector
Cloning vectors usually carry?
1) Selectable markers
2) Reporter Gene
3) Multi-cloning site
4) Origin of replication
Define: Selectable marker
Grow only in cells with plasmid is media is medicated
Indication of plasmid in cell
Define: Reporter gene
Blue/white screening
White positive for DNA inserted into gene
Indication of DNA inserted/cloned into plasmid
Define: Multi-cloning site
Restriction enzyme sites that will disrupt reporter gene
Allows for easy insertion of gene
Define: Origin of replication
Replicate DNA with high copy number
DNA of interest cut with?
What does it leave on DNA?
Restriction enzymes
Sticky ends on DNA
What enzyme repairs sticky ends on DNA strands?
DNA Ligase
How is cDNA made
Copying mRNA and reverse transcriptase it to cDNA
Uses for cDNA?
1) Exome sequencing
2) Cloning genes without introns in bacteria
3) Study gene expression (stability of mRNA; splice site variant; expression levels of mRNA)
4) Hybridization probe
5) Gene therapy
Steps in PCR:
1) Denature DNA
2) Cool down and add annealing primers
3) Extend primers with DNA polymerase II
4) Continue to repeat until enough copies of DNA present
Uses of PCR to amplify DNA:
Used when tiny amount of DNA available
Just need on molecule to amplify
Uses of PCR to Genetic testing:
Pre-natal testing
IVF
Paternity
Ancient DNA from fossils
Genealogy
Uses of PCR to make probes:
Easily labelled
Quick to produce
Uses of PCR to map out genetic changes:
Detection of single nucleotide polymorphism
Detect restriction fragment length polymorphism
ID pathogens
Types of probes used in DNA tech:
PCR
cDNA
Oligonucleotides
PCR as a probe type
Ranges from 50 bp to 2kb
DNA amplified to millions of copies
cDNA as a probe type
Segments of 50 bp 10 kb DNA
Oligonucleotides as a probe type
Synthetic DNA from 2bp to 100 bp
Types of Labelling in DNA Tech
Radioactive probes
Fluorescent probes
Oligonucleotides
Radioactive probe labels
Radioactive phosphorus on 5’ end
All molecules will look the same with 5’ radiolabelled
Fluorescent probe labels
Linked to chemicals that fluoresce
Able to use several probes of different colors at the same time
Oligonucleotide labels
Label added during production or after production
Restriction endonucleases
AKA Restriction enzymes
Method of action for restriction enzymes
Cut sequence-specific DNA sequences
Primarily made of?
dsDNA
Restriction enzymes bind to
Palindromic sequences in DNA
If palindromic sequence in DNA mutated - what becomes of restriction enzymes?
They are unable to cut sequence DNA since palindromic sequence is mutated
Gain or loss of restriction sites can be exploited in what way?
Analysis of Restriction Fragment Length Polymorphism
Bacteria will protect their DNA how?
Methylation
Restriction enzyme TaqI
Formulated from Thermus aquaticus
Recognizes T/CGA sequences to create sticky ends on fragments
Restriction enzyme HaeIII
Formulated from Haemophilus aegyptius bacteria
Recognizes 5’-GG/CC-3’ sequences to create blunt ends on fragments
Dot Blotting
DNA/RNA fixed to membrane then probe added to detect sequence
ONLY complementary DNA/RNA sticks to blot
Dot blotting useful for?
Drug response on expression of genetic markers
Classification of tumors in cancers
Gene expression levels in different cells
Show progression of disease
Localization of tissue specific expression
Variable Number Tandem Repeats
Occur in clusters with number of repeats varying in people
Makes use of microsatellites/repetative DNA
Restriction Fragment Length Polymorphism takes advantage of
Mutational changes
Mutations affect RFLP in what ways?
1) Longer/shorted fragments
Insertions yield longer fragments
Deletions yield shorter fragments
Point mutation NOT SEEN unless form/delete restriction site
RFLP used to show?
Fragment has been cloned as expected
DNA fingerprinting
Genome mapping
Disease detection
RFLP and Electrophoresis:
Use of agrose gel?
Use of Southern blotting?
Agarose gel only if DNA sample pure with few fragments
Southern blotting AFTER gel if whole genome needed or too many fragements
Microarrays in diagnostic testing
Detected viral strains, bacterial strains, drug resistance, and forensic contact
Genomic Wide Association Studies are used for?
- Find predisposing genes in multivariant diseases
*Find genes that are either upregulated that shouldn’t be or down regulated that shouldn’t be
Comparative Staging Expression Microarrays
More likely to be used for staging cancers via gene expression
*Detect normal, early or late staging
Screening Microarrays
ID Pathogens
Disease typing
Genotyping
Detect mutant/variant type DNA
Gene expression microarray
Amount of mRNA, timing of expression and splice variants in DNA
Single nucleutide polymorphisms
Detection on single point mutation in DNA
Describe interphase chromosomes
Not condensed
Barr body is only consolidation of genetic material
Barr body
Inactivated X chromosome that condenses down
Found evidence of the Barr body
Dr. Lyons
Structure of a chromatid
Centromere
p arm (shorted)
q arm (longer)
Telomeres at very end
Which phase of cell cycle to we arrest chromosomes to look at?
Metaphase
Karyotyping
Ordered display of all chromosomes from ONE cell
How are chromosomes ordered in karyotyping?
By size and/or position of centromere
Classification of this chromosome?
Metacentric
Classification of this chromosome?
Acrocentric
Classification of this chromosome?
Submetacentric
Chromosome numbers associated with acrocentric chromosomes?
13, 14, 15, 21, 22 & Y
Why are chromosomes 13, 14, 15, 21, 22, and Y so important?
Involved in Robertsonian translocation
Assignment of bands to chromosomes
Begin a centromere and work out
First number is REGION of chromosome
Second number is BAND ON chromosome in THAT region
Features of Dark G bands on chromosomes?
Later replication
Fewer transcriptionally active genes
More condensed
Higher AT base pairs
Define: Polyploidy
Multiple copies of chromosomes (usually 3)
Not viable with life
Triploid
Define: Trisomy
Excess of one extra chromosome
Only ones viable with life - 13, 18, 21
Written as (47, XX, +21)
Define: Monosomy
Lack of one chromosome
Monosomy X compatible with life
(45, X) / (45, XY, -16)
Trisomy and monosomy due to
Non-disjuction in meiosis I
Define: aneuploid
Wrong number of chromosomes
Define: euploid
Correct number of chromosomes
Maternal Age Effect:
Older a female gets, greater risk for non-disjuction events to occur
Eggs have been stuck in Prophase I since birth - that’s a long time to sit and stew.
Only trisomy with no affects from maternal age?
Trisomy 16
Describe sections of karyotype nomenclature
Number = number of chromosomes
XX/XY = sex chromosomes present
inv/del/dup/ins/r/t/rob/i
Number of chromosome involved
(Breakpoint in abnormality)
Balanced chromosomes
Nothing gained/lost
Unbalanced chromosomes
Too much gained or too much lost
Duplication (dup)
Describe events within one PAIR of chromosomes
Having 2 copies of same chromosomal segment
Insertion (ins)
Section of chromosome is copied and inserted into different chromosome
Recip chromosome mentioned first; donor chromosome mentioned second
Deletion/Insertion combo
Section excised from one place and inserted somewhere else
Isochromosome Formation (i)
Chromosome with 2 identical arms
Due to misattachment of spindle apparatus - separated from top to bottom, not side to side
Ring (r)
Any chromosome that loses telomeres at BOTh ends will become a ring structure
Causes instability of cell - DNA unable to replicate ring structure
Seen more often in cancer cells
Define reciprocal translocation (t)
Exchange of 2 acentric fragments of chromosomes
Breaks within arms that switch places with one another
Define Robertsonian translocation (rob)
Usually occurring in the acrocentric arms of chromosomes (13, 14, 15, 21, 22, Y)
Satellites break off at ends of chromosomes and centromeres fuse together
Common cause of losing a chromosome (46 -> 45)
Results of Reciprocal translocations:
Every case is phenotypically different
Developmental defects ARE common
Able to mimic known syndromes
Leads to multiple affected tissues (heart)
Results of Robertsonian translocations
Genes in satellite are found in SEVERAL places
No symptoms in balanced form
Philadelphia chromosome
Short chromosome containing centromere of 22 and tip of q-arm of 9
Product of reciprocal translocation
Joins chromosomes 9 and 22 together
