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American Society of Cytopathology Core Curriculum in Molecular Biology Copyright 2010 American Society of Cytopathology
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American Society of Cytopathology Core Curriculum in Molecular Biology
Chapter 2
Molecular Science
Nucleic Acid Biochemistry
Stephanie A. Hamilton, EdD, SCT, MB(ASCP)CM
MD Anderson Cancer Center
Houston, Texas
Copyright 2010 American Society of Cytopathology
Molecular Pathology
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• Molecular pathology is the study of nucleic acids (DNA and RNA) from patient specimens mainly to diagnose genetic diseases, direct choice of therapy, detect presence of pathogens and for identity testing
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Genome and Cells
• Each organism has a unique genome
• A genome includes all of the genes of the organism
• All cells in a person’s body carry the same genome or genetic information in the nucleus. The expression of different genes is what determines cell function thus producing a nerve cell, muscle cell or skin cell
Copyright 2010 American Society of Cytopathology
Cell, Chromosome and DNA
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http://members.cox.net/chromosome3/ Accessed 10/19/2010
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Chromosomes, Genes and DNA
• A gene can be defined as a region of DNA that controls a hereditary characteristic
• Genes are a sequence of nucleotides in DNA helix
• Human genome includes all of the genes (about 30,000 genes) located on 23 pairs of chromosomes
• There are 22 pairs of autosomes and 1 pair of sex chromosomes, XX or XY • The human genome contains about 3 billion nucleotide pairs
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Genetic Information
• Genotype: the DNA nucleotide sequence responsible for a phenotype
• Phenotype: a trait or group of traits resulting from transcription and translation of genes
• Position effect: A gene inserted or moved into a different chromosomal location, it may be expressed differently than it was in its original position
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Watson-Crick Model
Crick-Crick Model
• In 1953, James Watson and Francis Crick proposed structure for DNA • Watson‐Crick model: a DNA molecule consists of two polynucleotide strands coiled around each other in a helical "twisted ladder" structure
• Two sugar‐phosphate backbones are on the outside of the double helix forming the two sides of the ladder • Bases are on the inside forming the rungs of the ladder. • Bases follow a specific base pairing rule: Adenine pairing with Thymine and Guanine pairing with Cytosine
• How the bases are arranged in the DNA determines the genetic code Copyright 2010 American Society of Cytopathology
Nucleic Acids
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• The nucleic acids are polynucleotide chains since the units from which nucleic acids are constructed are called nucleotides.
• Each nucleotide consists of three components: – a nitrogenous base, either a purine or a pyrimidine
– a pentose sugar, deoxyribose (DNA) or ribose (RNA)
– a phosphate group
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Nucleotides
• A nucleotide may have one of more phosphate groups
– Example: adenosine with one phosphate is adenosine monophosphate (AMP); adenosine with 3 phosphates is adenosine triphosphate (ATP)
• Nucleotides can be converted to nucleosides by hydrolysis
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Bases
• The nitrogenous bases are of two kinds:
purines and pyrimidines
• Purines: – Two ring structures: fused five (pentamer)‐ and six (hexamer)‐membered rings
• Adenine (amino purine)
• Guanine (amino‐oxypurine)
• Pyrimidines: – Single six‐membered ring structures
• Cytosine (oxy‐aminopyrimidine)
• Uracil (di‐oxypyrimidine) • Thymine (di‐oxy‐methylpyrimidine)
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Bases in DNA and RNA
In DNA
2 oxy groups (ketone groups)
Replaces
thymine in RNA
Pyrimindines
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Purines
Nitrogenous Bases in DNA
NH2
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Double bond O
NH2 group
CH3 methyl group
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Nucleotide and Nucleoside
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• A base linked to a sugar and a phosphate is called a nucleotide
– Ex. ATP (adenosine triphosphate); ADP (adenosine diphosphate)—energy is released when phosphates are removed
• A base linked to a sugar is called a nucleoside
– Attached sugar may be ribose (in ribonucleic acid or RNA)
– Attached sugar may be deoxyribose in deoxyribose acid or DNA) Copyright 2010 American Society of Cytopathology
Sugars in DNA and RNA
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• Both DNA and RNA have five‐carbon sugar
• Deoxyribose in DNA or Ribose in RNA
• Deoxyribose:
– a monosaccharide containing five carbon atoms
– includes an aldehyde functional group in its linear structure
– it is a deoxy sugar derived from the pentose sugar ribose by the replacement of the hydroxyl group at the 2 position with hydrogen, leading to the net loss of an oxygen atom. Copyright 2010 American Society of Cytopathology
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Ribose and Deoxyribose Sugars
Has one oxygen atom
less; thus “deoxy”
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What are the differences between
DNA and RNA?
DNA
RNA
Base: Thymine
Base: Uracil
Sugar: Deoxyribose
Sugar: Ribose
Strands: Double stranded
Strands: Single stranded
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How are the three subunits linked?
• The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms in the sugar rings. • The point of attachment of the sugar to the base is the hydroxyl group on C‐1’ carbon atom.
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Each nucleotide consists of a five-carbon sugar, the first carbon of which is covalently joined to
a nitrogen base and the fifth carbon to a triphosphate moiety
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DNA Complementary Base Pairing
• Each type of base on one strand forms a bond with just one type of base on the other strand, called complementary base pairing
• Purines form hydrogen bonds to pyrimidines
– A bonding only to T
– C bonding only to G
• AT and GC are called base pairs
• How the bases are arranged in the DNA determines the genetic code
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Structure of DNA
• In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand
• The asymmetric ends of a strand of DNA bases are referred to as the 5’ (five prime) and 3’(three prime) ends
• The DNA double helix is held together by hydrogen bonds between the bases of the two strands
– AT forms two hydrogen bonds – GC forms three hydrogen bonds
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Hydrogen Bonds Between Bases
• Both the percentage of GC base pairs and overall length of a DNA helix determine the strength of the association between the two strands
– Double‐stranded sequences rich in G and C hold together more strongly (thus, have a higher “melting point”) than sequences rich in A and T
• Hydrogen bonds are not covalent, so they can be broken and rejoined relatively easily
• The two strands of DNA can therefore be pulled apart like a zipper, either by a mechanical force or high temperature
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DNA Structure
http://www.bae.uky.edu/~snokes/BAE549thermo/microbio/nucleicacids.htm. Accessed 6/23/10
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DNA Double Helix
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Major groove
Minor groove
• As a result of asymmetries in base pairing, longer antiparallel DNA double strands will begin to twist
• Appears to have alternating wide and narrow grooves called “major” and “minor” grooves
• There are different 3‐D forms of DNA
Buckingham, L and Flaws, ML. Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications. Philadelphia: F.A. Davis
Company, 2007.
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Forms of DNA Structure
• B form
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Twists to the right, clockwise
Helix makes a turn every 3.4 nm
Neighboring base pair is 0.34 nm
Intertwined strands make 2 groove based on widths
Has major and minor grooves
Usually found in vivo or in solution
• A form
– Twists to the right
– Turns at 2.3 nm
– Exists in alcohol or salt solution (dehydrated solutions)
• Z form
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Twists to the left
Turns at 4.6 nm
Exists in high salt or alcohol solution
Has high CG contents
Still controversial if it does exist in living cells
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Units of Measurement
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• The following abbreviations are commonly used to describe the length of a DNA molecule:
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bp = base pair(s) kb (= kbp) = kilo base pairs = 1,000 bp mb = mega base pairs = 1,000,000 bp gb = giga base pairs = 1,000,000,000 bp • When DNA is single stranded or in RNA the measurement is in number of nucleotides‐nt (base pair, pentose sugar & phosphate group)
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Chromosomes
• A gamete has 1 copy of the genome, and is called haploid
• Diploid organisms, inherit a haploid set of all their genes (23 chromosomes) from each parent; thus, humans have 2 copies of every gene except X & Y chromosomes • Euploid: Cell with a normal complement of chromosomes
• Aneuploid: Cell with an abnormal complement of chromosomes (such as an increased number of chromosomes)
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http://picsdigger.com/image/a0bb3308/ Accessed 10/19/2010
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Chromosome
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• A chromosome has the following components:
– Centromere ‐ A constricted chromosome region to which spindle fibers attach during cell division
– Telomere ‐ The ends of linear chromosomes that are required for replication and stability
– p arm is the short arm of a chromosome
– q arm is the long arm of a chromosome
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Histones and Nonhistones
• If uncoiled, the DNA contained by each chromosome would be very long and difficult to fit into a cell
• Chromosomal DNA is packaged into a compact structure with the help of specialized proteins called histones
• The DNA plus histones together is called chromatin
• The DNA double helix wraps around a central core of eight histone protein molecules (an octamer) to form a single nucleosome
• The nonhistone protein molecules help the DNA molecule to condense further to fit into the chromosomes
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How the DNA is kept compact?
http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/lamar/histoneh1.htm Accessed 10/19/2010
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Histones
• Five types of histones: – H1 (H5), H2A, H2B, H3, H4
• H1 and its homologous protein H5 are involved in higher order structures
• Other 4 types of histones along with DNA forms nucleosomes
• Each nucleosome consists of 146 bp DNA and 8 histones (2 pairs of each)
• DNA is wrapped around the histone core
• These histones contain lysine residues which provide a positive charge—which interacts well with the negative charged DNA creating a opposite bond
• One way to weaken this bond is through acetylation. This may neutralize the positive charge and weaken the binding force between histones and DNA (This step does occur during transcription)
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Heterochromatin and Euchromatin
• Heterochromatin: –
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Tightly coiled, highly‐compacted DNA
Less available for RNA transcription
Genetically inactive
Found only in eukaryotic (with nucleus) cells
• Euchromatin:
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Lightly packed, more open form of chromatin
DNA not bound to a histone complex
Rich in gene concentration
Often under active transcription
Found in eukaryotes and prokaryotes (cells without nuclei)
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Extrachromosomal Structures:
Bacteriophage
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Bacteriophage genomes are mosaic
Genome of any one phage species appears to be composed of numerous individual modules
These modules may be found in other phage species in different arrangements
Mycobacteriophages, bacteriophages with mycobacterial hosts, are excellent examples of mosaicism
http://en.wikipedia.org/wiki/Bacteriophage#Genome_structure
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Extrachromosomal Structures:
Plasmids
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Plasmids are:
– Extra circular genetic that can be passed from bacteria to bacteria (bacterial conjugation)
– In biotechnology, plasmids are often used in recombination work
• Some other organism’s gene is inserted into bacteria plasmid
• Then, bacterial multiply and transcribe inserted gene into many useful products (vector)
http://wiki.answers.com/Q/What_is_the_function_of_plasmid
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Example of Plasmid: Ti stands for Tumor inducing
http://arabidopsis.info/students/paaras/t_dna.htm
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Extrachromosomal Structures:
Mitochondrial
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Mitochondrial DNA (mt DNA or mDNA)
Circular, covalently closed, double‐
stranded
DNA located in mitochondria
Converts the chemical energy from food into ATP
Can be regarded as smallest chromosome (15,000‐17,000 bp
Is inherited solely from mother
Derived from circular genomes of bacteria that were engulfed by eukaryotic cells (endosymbiotic theory)
Each mitochondrion estimated to contain 2‐10 mtDNA copies
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Extrachromosomal Structures: Yeast
Artificial Chromosomes (YACs)
• YACs are vectors
• Used to clone DNA fragments larger than 100 kb and up to 3000 kb
• Useful for cloning large genes and for mapping complex genomes
http://www.accessexcellence.org/RC/VL/GG/YAC.php
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http://en.wikipedia.org/wiki/Yeast_artificial_chromosome
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Mass Spectrometry
• Is an analytical technique
• Measures the mass‐to‐charge ratio of charged particles
• Used to determine:
– masses of particles – Elemental composition of a sample or molecule
– Chemical structures of molecules such as peptides and other chemical compounds
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Mass Spectrometry
• How it works:
– Ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass‐to‐charge ratios
• MS instruments consist of:
– An ion source
– A mass analyzer (sorts ions by their masses by applying electromagnetic fields)
– A detector (measures the value of an indicator quantity)
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