Genetics Block One

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<p>Part One<br /> Lectures 1-7</p>

Chromosome disorders

Gain or loss of genes in whole chromosomes or segments of chromosomes

e.g. extra copy of chromosome 21 results in Down syndrome

DNA

DNA structure carries the chemical information for exact transmission of genetic information from one cell to its daughter cells and from one generation to the next. Composed of: a five carbon deoxyribose sugar; a nitrogen-containing purine or pyrimidine base; and a phosphate group.

DNA primary structure (sequence of C's, A's, G's, + T's) specifies the amino acid sequences of the polypeptide chains of proteins

Gene

maps the chromosomal location of the genes

Karyotype

species specific characteristic chromosome complement (number and morphology)

Cytogenetics

study of chromosomal structure and inheritance

Somatic cells

all cells in the body except those of gamete-forming germline most are diploid

Alleles

one of the alternate versions of a gene or DNA sequence at a given locus

Purines

adenine (A) and guanine (G)

Pyrimidines

thymine (T) and cytosine (C)

Nucleotide

base, phosphate, and sugar polymerized into long polynucleotide chains by 5'-3' phosphodiester bonds formed between
adjacent sugar units forming a double helix

What is the largest and smallest chromosome?

Chromosome 1-largest with 250 million base pairs

Chromosome 21-smallest with 50 million base pairs

If guanine makes up 23% of the nucleotides in a sample of DNA then thymine would make up what percent of the bases?

27%

A sample of human DNA is subjected to increasing temperature until the major fraction exhibits optical density changes due to disruption of its helix (melting or denaturing). A smaller fraction is atypical in that it requires a much higher temperature for melting. This smaller, atypical fraction of DNA must contain a higher content of which bases?

Cytosine and Guanine

Which of the following is a substrate for RNA synthesis, but not DNA synthesis?

adenosine triphosphate (ATP)

Describe Mitochondrial chromosome

important part of human genome. Have their own genetic code and Replication machinery.

In most multicellular organisms, mtDNA is maternally inherited.

In the mitochondria, DNA is in the form of plasmids or circular loops of DNA

Describe the bonds between DNA bases?

A-T BONDS have 2 hydrogen bonds

G-C BONDS HAVE 3 hydrogen bonds and have a high melting temperature

chromatin

The complex of DNA and proteins that makes up a eukaryotic chromosome. When the cell is not dividing, chromatin exists as a mass of very long, thin fibers that are not visible with a light microscope. There are two types of chromatin: euchromatin & heterochromatin. There are four core histones with two copies each of H2A, H2B, H3, and H4 constitute an octomer.

euchromatin

A chromosome region that stains poorly (light band) and genetically active part of chromatin transcribing genes. Region of eukaryotic chromosome that is not fully condensed in the interphase nucleus.

heterochromatin

Nontranscribed eukaryotic chromatin that is so highly compacted that it is visible with a light microscope (dark band) during interphase. Consists mostly of repetitive DNA that shows little or no genetic activity

Four core histones

two copies each of H2A, H2B, H3, and H4 constitute an octomer

Nucleosome

basic structural unit of chromatin & fibers appear as "beads on a string"

Describe the roles of H1, H3, H2A, H4

140 bp segment of DNA wound around each octomer just under two turns forming the nucleosome (beads on string)

H1 BINDS DNA AT EDGE OF EACH NUCLEOSOME (LINKER). Contributes to solenoid 30nm fiber

H3 and H2A CONFER SPECIFIC characteristics on the genomic DNA

H3 and H4 can be POST TRANS MODIFIED

Histone code

can vary between cell types to regulate gene expression

Solenoid

a secondary helical chromatin structure of coiled nucleosomes appear as a thick 30-nm-diameter cylindrical fiber

Solenoids packed into chromosome loops attached at 100 kb intervals to a protein scaffold

Interphase of cell cycle chromosomes in most decondensed state

The major function of histone proteins is to..?
A) Decondense chromatin
B) Package DNA
C) Stabilize the DNA helix
D) Unwind DNA
E) Regulate transcription

B) Package DNA

Single-copy DNA sequences

one-half of genome function a mystery

Most in short stretches (several kb or less) interspersed with various repetitive DNA families

Repetitive DNA sequences

Repetitive DNA sequences repeats clustered in one or a few locations or interspersed throughout the genome. Different types of tandem repeats are called satellite DNAs and can be separated as a distinct fraction of DNA.

Tandem repeat families

Vary in their location; total length of tandem array; and length of repeat units in the array.

Molecular tools for clinical cytogenetic analysis due to ease of detection.

Pentanucleotide repeats

long arrays found in genetically inert regions on chromosomes 1, 9, and 16 and more than one-half of Y chromosome

alpha-satellite family

171 bp unit repeat at the centromere of each chromosome attached to microtubules of the spindle apparatus

Alu family

best studied repetitive element related sequences. 300 bp in length dispersed throughout the genome. Implicated in genetic diseases the more than a million Alu family members are at least 10% of the human genome.

LINE

Long interspersed nuclear element family or L1 that is up to 6 kb long and about 850,000 copies or ~20% of the genome. Repetitive DNA can cause mutations in a gene. Copies can be integrated to cause insertional inactivation. Can be duplicated (often with extraordinary high sequence conservation)

Segmental duplications

substantial segments of a chromosome these genomic rearrangements can lead to gene deletions resulting in disease. Usually misalign in meiosis I.

Deletions often appear to be the result of unequal crossing over of homologous chromosomes during meiosis. What promotes unequal crossing over events and, hence, deletions?

Alu repeat sequence elements

Name the three types of DNA

Single Copy DNA 45%: function remains a mystery as only 1.5% encodes proteins

Dispersed Repetive DNA 45% -> Maintainence of chromosomal structure

Satellite DNA 10% -> Different types of tandem repeats

Two patients with hemophilia A have been found in which the disease is due to the de novo insertion of a retrotransposon L1 LINE into exon 14 of the factor VIII gene during embryogenesis or maternal oogenesis. Although L1 LINEs are common within the human genome, almost all of those carried in humans today are unable to move. What is most likely a characteristic of the rare insertion that created hemophilia in these two patients?

RNA polymerase II activity was required for LINE sequence duplication and insertion

Mitosis

A process of nuclear division in eukaryotic cells conventionally divided into five stages: prophase, prometaphase, metaphase, anaphase, and telophase. Mitosis conserves chromosome number by equally allocating replicated chromosomes to each of the daughter nuclei.
Ordinary somatic cell division results in 2 diploid daughter cells with the 2n chromosome complement of 46 chromosomes identical to the parent cell.

Chromosome spread

condensed chromosomes (sister chromatids) visible during metaphase or prometaphase

Centromere

primary constriction due to formation of kinetochore, divides chromosome into two arms, short arm (p) & long arm (q).

Microtubule organizing center.

Telomeres

ends of each chromatid; specialized repetitive DNA sequences that ensure chromosome integrity during cell division.

Telomerase

enzyme that maintains chromosome ends

5 Stages of Mitosis

1. Prophase
2. Prometaphase
3. Metaphase
4. Anaphase
5. Telophase
*Cytokinesis

Centrosomes

A pair of microtubule organizing centers that form foci from which microtubules radiate and gradually move to opposite poles of the cell.

Congression

chromosomes move toward point midway between spindle poles.

Anaphase

Begins abruptly as chromosomes separate at centromere, sister chromatids now independent daughter chromosomes and move to opposite poles

Cytokinesis

process where cytoplasm is cleaved to complete cell division. Begins as chromosomes approach spindle poles and results in two daughter cells with all the genetic information.

Prophase

initiates mitosis, marked by chromosomal condensation and formation of the mitotic spindle

Metaphase

maximal condensation and arrangement at the equatorial plane of the cell

Telophase

chromosomes begin to decondense and nuclear membrane reforms around each daughter nuclei

Prometaphase

nuclear membrane breaks up; chromosomes disperse and attach to microtubules by kinetochores and continue to condense

DNA synthesis in S phase

Chromosomes replicate to become bipartite with two sister chromatids

The protein complex which assocates with the centromere sequence on the chromosome is called the..?

Kinetochore

The longitudinal halves of a chromosome that has undergone replication are known as...?

Chromatids

After S phase a chromosome exists as...?

a pair of sister chromatids each with its own centromere

A medical student is studying a liver biopsy taken from a regenerating liver following a partial hepatectomy. In one dividing cell, the chromosomes are aligned in the center of the cell. What is best describes the character of the chromosomes aligned in that cell?

92 chromatids creating 46 chromosomes with kinetochore microtubules from both spindle poles attached to each centromere

Chiasmata

a point of overlap of paired chromatids at which fusion and exchange of genetic material occurs during prophase I of meiosis

The diploid human genome is organized into _____________ , _______________ chromosomal DNA molecules:

46, linear

When are eukaryotic chromosomes highly packaged?

During cell division

What is G-banding?

staining chromosomes with Giemsa stain which is technique most widely used in clinical cytogenetics labs. Chromosome pairs stain with characteristic pattern of alternating light (euchromatin) and dark (heterochromatin) G bands. Reflects base composition and distribution of repetitive DNA elements

A G-band segment of metaphase chromosomal material appears lighter than surrounding areas. A fluorescence in situ hybridization probe for a known transcribed gene binds in the same region. This segment most likely consists of which of the following?

Euchromatin

Karyotyping

cutting chromosomes out from a photomicrograph and arranging them in pairs

Meiosis

occurs in germs cell and results information of reproductive cell gametes with only 23 chromosomes and one of each autosome.

Consists of 1 round of DNA synthesis and 2 rounds of chromosome segregation and cell division the successive meiotic divisions are Meiosis I and Meiosis II.

Meiosis I

chromosome number reduced by half and genetic recombination (meiotic crossing over) occurs as homologous DNA segments are exchanged between non-sister chromatids of a pair of homologous chromosomes which ensures no gametes are identical.

Meiosis II

follows meiosis I without intervening DNA replication and chromatids separate and 1 chromatid of each chromosome passes to each daughter cell.

2nd female arrest point at Anaphase II.

What are the phases of Meiosis I

Prophase (Leptotene, Zygotene, Pachytene, Diplotene, Diakinesis)

Metaphase I

Anaphase I

Telophase I

Cytokinesis

Describe Meiosis and its consequences

Reduction of chromosome number from diploid to haploid, essential step in the formation of gametes.

Segregation of alleles, at meiosis I or meiosis II, in accordance with Mendel's first law. Shuffling of genetic material by random assortment of the homologues, in accordance with Mendel's second law.

Crossing Over: allows for 2^23 possible genetic variations)

The kinetochore is found associated with which of the following DNA sequences on the chromosome?

The centromere

Describe the daughter cell during the telophase I of the meiotic stage of the human ovum.

Each daughter cell contains 23 chromosomes. Each chromosome at this stage is composed of two sister chromatids, which are similar but not always genetically identical.

How many oocytes are produced in the female meiotic division?

In the female, the two meiotic divisions produce only one oocyte.

What are the phases of Prophase I in Meiosis I

Prophase I-complicated process with multiple defined stages (Led Zeppelin Probably Did Drugs)

Leptotene
Zygotene
Pachytene-(stage with meiotic crossing-over)
Diplotene - (first female arrest point)
Diakinesis

Leptotene

chromosomes replicated in preceding S phase become visible as thin threads and two sister chromatids align and cannot be distinguished

Zygotene

homologous chromosomes align, synapsis held together by synaptonemal complex essential for recombination

Pachytene

chromosomes much more tightly coiled and synapsis complete and homologue pairs appear as a bivalent (tetrad of four chromatids) and meiotic crossing-over occurs

Diplotene

after recombination the synaptonemal complex breaks down and bivalents separate. Two homologues of each bivalent are held together at chiasmata (mark cross-overs). Diplotene is the first arrest point in females which occurs at 5 months into fetal life. 2nd arrest point at Anaphase II.

Diakinesis

maximal condensation

Stages of Spermatogenesis

The sequence of events begins at puberty and yields 4 Sperm. It takes about 64 days in humans.

Sperm formed in seminiferous tubules of testes. Last cell type in developmental sequence is primary spermatocyte undergoes meiosis I to form two haploid secondary spermatocytes Rapidly undergo meiosis II, the two secondary spermatocytes form four spermatids that differentiate without further division into sperm.

Oogenesis

Primary oocytes nearly all complete prophase I by birth, those that do not degenerate remain arrested in prophase I for years, until ovulation during the menstrual cycle at sexual maturity, individual follicles grow and mature, and a few (~one per month) are ovulated.

Before ovulation, the oocyte rapidly completes meiosis I, dividing to become a secondary oocyte and first polar body. Meiosis II proceeds to metaphase and is completed only if fertilization occurs.

Single gene defects

Individual mutant genes are rare but the ones that do show obvious and characteristic pedigree patterns.

Mutation present on one or both chromosomes of the pair. ex. Cystic fibrosis, sickle cell anemia, and Marfan syndrome

Multifactorial disorders

result from complex interactions between genetic and environmental factors. Have genetic contribution but may not show a mendelian inheritance pattern in families.

Responsible for majority of diseases ex. prenatal developmental disorders, Alzheimer disease, diabetes, and hypertension

Metaphase I

Nuclear membrane disappears, spindle forms and paired chromosomes align on equatorial plane with centromeres oriented toward different poles

Anaphase I

Two members of each bivalent move apart and undergo disjunction as their respective centromeres with attached sister chromatids are drawn to opposite poles. The chromosome number is halved and each cell has haploid chromosome number.

Nondisjunction

Pathogenic process when both homologues of a chromosome pair travel to the same pole

Telophase I

Two Haploid sets of chromosomes grouped at opposite poles

Cytokinesis prior to Meiosis

Cell divides into haploid daughter cells and enters brief meiotic interphase. Meiosis II begins with no S phase between meiotic divisions as with mitotic interphase.

Describe Human oogenesis and
fertilization in relation to
the two meiotic divisions.

The primary oocytes are formed prenatally and remain suspended in prophase of meiosis I for years until the onset of puberty. An oocyte completes meiosis I as its follicle matures, resulting in a secondary oocyte and the first polar body. After ovulation, each oocyte continues to metaphase of meiosis II. Meiosis II is completed only if fertilization occurs, resulting in a fertilized mature ovum and the second polar body.

Genetic code

genetic information stored in DNA as the sequence of adjacent DNA bases determines sequence of amino acids

RNA splicing

removal of introns and splicing together of exons.

Mutations in the GT at 5' or AG at 3' impair RNA splicing and reduce the amount of normal mature b-globin mRNA. This is observed in b-thalassemia patients.

AATAAA

3' UTR - contains AATAAA signal for polyadenylation.

Introns

removal of introns and splicing together of exons. Noncoding regions of DNA sequence that lie between coding regions, removed through DNA splicing.

Exons

a coding region of a eukaryotic gene; when expressed, they are separated from each other by introns

Transcription factors

interact with specific DNA sequences to determine the spatial and temporal pattern of gene expression

Pseudogenes and name the 2 types

DNA sequences closely resemble known genes but are nonfunctional. Found in both the b-globin and OR gene families. Pseudogenes widespread in the genome and have two general types:

1. Nonprocessed pseudogenes nonfunctional "dead" genes

2. Processed pseudogenes arise from retrotransposition

microRNA (miRNA)

important class of noncoding RNAs. Short 22 nt long RNAs which control expression or repression of other genes during development

coding or sense strand

5' to 3' strand of nontranscribed DNA is the coding or sense strand

noncoding or antisense strand

3' to 5' transcribed template strand is the noncoding or antisense strand

Transcription of DNA to mRNA

Protein coding genes transcribed by RNA polymerase II. 5' to 3' strand of nontranscribed DNA is the coding or sense strand. 3' to 5' transcribed template strand is the noncoding or antisense strand. Primary RNA transcript is processed by the addition of a "cap" structure at the 5' end and cleavage at the 3' end. A poly(A) tail of is added to the 3' end of the RNA. These post-transcriptional modifications are done in the nucleus.

Central Dogma

DNA (in the nucleus) → RNA → Proteins (in the Ribosomes)

Transcription: Process of making mRNA from DNA

Translation: Process of making polypeptide chains from mRNA in the Ribosomes via tRNA

Codon

3 adjacent bases along the mRNA specific for a particular A.A.

At any position 4 possibilities of A, U, C, or G so for 3 bases 43 or 64 possible triplet combinations constitute the genetic code

Only 20 A.A.s and 64 possible codons and most A.A.s are specified by more than 1 codon making it a degenerate code

AUG

AUG is always the initiation codon so methionine is the first encoded (amino-terminal) amino acid

AUG establishes the reading frame of the mRNA

Describe Transcription of the Mitochondrial Genome

The mitochondrial genome has distinct transcription and protein-synthesis systems.

A specialized RNA polymerase (encoded in the nuclear genome) transcribes the entire genome using 2 related promoter regions one on each strand of the circular genome.

Transcripts are then processed to generate the various individual mRNAs, tRNAs, and rRNAs.

b-globin

Occupies 1.6 kb on the short arm (p) of chr.11 and has 3 exons and 2 introns transcribed in a centromere in the telomere direction.

Mutations in either element causes a sharp reduction in transcription leading to B-Thallassemia.

b-globin promoter

short functional elements interact with specific transcription factors which includes proteins that restrict expression to hemoglobin producing erythroid cells.

TATA box

conserved region rich in A's and T's 25 to 30 bp upstream of start site which determines start of transcription for b-globin mRNA and includes 50 bp of 5' UTR upstream of the AUG initiation codon

CAT box-(CCAAT)

second conserved region upstream of TATA box

Housekeeping genes

Constituively expressed in most or all tissues and often lack CAT/TATA Boxes. Promoters are often CG-rich and located in regions of the genome called: CpG islands (high in 5'-CG-3'). CpG islands can be modified by the addition of a methyl group.

Methylation of DNA is associated with repression or silencing. Hypermethylation results in Fragile X Syndrome. Gene inactivation is important in cancer, development, genomic imprinting, and X chromosome inactivation.

Enhancers

"activating" sequence elements act at a distance to stimulate transcription. The b-globin gene has tissue-specific enhancers present in the gene and in flanking regions

Locus control region (LCR)

required for normal expression of b-globin during development and distant sequences located upstream of the e-globin gene which establish proper chromatin context for high-level expression.

Introns within primary eukaryotic transcripts

A) Often function as exons in other genes

B) Are highly conserved in nucleotide sequence

C) Vary considerably in size and number among different genes

D) Are joined to form mature mRNA

C) Vary considerably in size and number among different genes

The DNA of gene X in humans has been isolated, cloned, and sequenced and is 8000 base pairs in length. From cells that were actively transcribing gene X, a cDNA library was made. In this case,
gene X was found to be transcribed from a mRNA that was 4200 bases in length. The most probable explanation of these results is that..

the DNA encoded introns that were excised during mRNA processing.

Alternative Splicing

The primary transcripts of one third of all human genes can be alternatively spliced, result in multiple mRNAs being translated into different proteins.

A potassium channel mutated in an inherited form of epilepsy has 35 exons, 8 are alternatively spliced leading to more than 500 different mRNAs each encoding a channel with different functional properties.

Polyadenylation

Mature B-globin mRNA has a 130 bp 3' UTR between the Poly-A-Tail and STOP codon.

Cleavage of the 3' end and poly-A is controlled by the AAUAAA sequence which is 20bp upstream of the Poly-A-tail.

B-Thallassemia patients observe a mutation in this region.

Gene Regulation

Activation or repression of gene expression involves changes in transcriptional control. Regulation not structure of the genome that changes dynamically. Ig Genes can somatically rearrange. Cutting and pasting of DNA sequences (V,D,J,C) to yield a highly variable antibody assortment. Occurs only in Ig and T-cell receptor gene clusters of B/T cell lineages.

Allelic Exclusion: Only 1 of 2 parental alleles of autosomal loci are expressed

Olfactory Receptor (OR) Genes

350 functional OR genes responsible for our acute sense of smell.

OR genes located throughout genome on nearly every chromosome; nearly half of OR Genes are located on c.11

OR Gene family is part of a much larger gene superfamily: G-protein coupled receptors

Retrotransposition

involves transcription, reverse transcription of a DNA copy of the mRNA, and the cDNA is copied back into the genome.

Fundamentals of gene expression

Initiation of transcription influenced by promoter regulator elements. Transcription Factors: interact with specific sequences to determine the spatial and temporal pattern of gene expression. Start site at beginning of the 5' UTR and continues several hundred to 1 million bp through introns and exons. 5' and 3' ends of mRNA are modified by a 7-methylguanosine cap and a poly-A tail.

RNA splicing-removal of introns and splicing together of exons.

Transcription

RNA Pol II reads DNA 3-5' and transcribes mRNA 5-3'.

Primary RNA transcript is capped at 5' and addition of Poly-A tail at 3' end. mRNA is then finally shipped out of the Nucleus.

Translation

mRNA translated into protein by tRNA's specifically bound to a particular AA. tRNA is 70 to 100 nts long and present the anticodon to the codon, which are the 3 adjacent bases along the mRNA sequence. Translation occurs on Ribosomes which are complexes made of 18S and 28S rRNAs and other ribosomal proteins. Since there are only 20 AA's, and 64 codons, most AA's are specified by more than 1 codon which makes it degenerate.

STOP codons

UGA, UAA, & UAG

What direction is proteins synthesis?

Proteins are synthesized from the amino (N) to carboxyl C terminus as the mRNA is translated 5-3' direction.

Two disorders associated with Beta-Globin Gene

Different mutations in the beta globin gene (3 exons) causes a variety of important disorders of Hb)

• Thallassemia
• Sickle Cell Disease

BRCA1 Gene

Mutations in the BRCA1 gene (24 exons) are responsible for many cases of inherited breast or breast and ovarian cancer. BRCA1/2 are ubiquititously expressed and repair dsDNA breaks via Homologous recombination. Tumor formation follows 2-hit hypothesis: both alleles of either BRCA1/2 lose function in tumor cells. The somatic LOF by the 2nd allele occurs by a loss of heterozygosity, mutation, or silencing due to methylation. The mutations tend to exhibit autosomal dominant inheritance patterns.

MYH7 Gene

Mutations in the beta-myosin heavy chain (MYH7) gene (40 exons) lead to inherited hypertrophic cardiomyopathy.

Allelic Exclusion

Only 1 of 2 parental alleles of autosomal loci are expressed in immunogenetics. Only 1 of the pair of alleles for each H chain and L chain for antibodies are expressed in a single cell.

Molecular Cloning

isolation of a particular gene or other DNA sequence in large quantities for further study.

Requires transfer of the DNA of interest into a single bacteria cell to grow in culture to amplify DNA and then isolate a large quantity
of recombinant DNA from the clone.

Restriction Enzymes

Originally isolated from bacteria. Recognize specific sequences in the DNA which cleaves palindrome sequences:
• Blunt Ends, 5', or 3' overhang (sticky ends) allow for the annealing of DNA making it easier to ligate the plamid strands once the clonal DNA has been inserted.
• EcoRI: 5'-GAATTC-3' always cut leaving a 4 base, single stranded 5'-AATT-3' overhang. Can join DNA in vitro by inserting DNA with complimentary overhang and then ligating with DNA ligase this yields a recombinant DNA molecule.

Palindromes

sequences of DNA that are identical when read from 5' to 3' direction on one strand and the 3' to 5' on the other strand.

Vector

DNA Molecule that replicates autonomously in bacterium or yeast for subsequent purification.

Common cloning vectors include plasmids, bacteriophage lambda, and bacterial artificial chromosomes (BACS).

Human DNA fragment inserted into a vector with DNA ligase transforms into bacterial host for propagation often with a high copy number per cell & grows up bacteria and purify large quantity of DNA.

Recombinant DNA Technology

Ligation of DNA molecules from different sources.

Plasmids

Small circular dsDNA; separate from host chromosome and independently replicate.

Vector plasmids in bacteria confer antibiotic resistance.

Plasmids have 3 critical components:
• Origin of replication
• 1 or more selectable markers
• 1 or more unique restriction sites for cloning foreign DNA

Bacterial Artificial Chromosome (BAC) vector:

specially designed for cloning large 100-350 kb inserts (made for large DNA inserts)

Libraries

collection of recombinant clones.

Vector has different DNA fragments derived from total DNA or RNA of a cell or tissue.

Genomic Libraries

DNA fragments of partial restriction digest via high frequency enzyme.

Collection of overlapped fragments which are big enough to clone in a BAC, and create a library.

Each of the resulting BAC clones at the bottom contains a different but partly overlapping fragment of human DNA. A collection of several tens of thousands of such BACs would represent all of the DNA from the human genome.

Complementary DNA (cDNA) Libraries

cDNA is synthesized with mRNA as a template using reverse transcriptase. Contains only exons and no introns or promoter sequences. cDNA set made from transcripts of a single gene may differ indicating alternate promoters or polyadenylation sites being used, or alternative splicing of coding exons.

You are attempting to clone your first gene. Up to the point of screening, everything has gone well. The vector has a gene for ampicillin resistance. In addition to the gene DNA, the insert also carries a gene for streptomycin resistance. Based on restriction digest, if properly inserted into the vector, your inserted DNA should be located within the ampicillin gene. Therefore, when screening you should look for clones that are..?

ampicillin sensitive and streptomycin resistant

Reverse Transcriptase

Enzyme used for cloning cDNA from mRNA.

cDNA (ss) can be used as a template for DNA polymerase to make a (ds) molecule which can be ligated into a vector for cloning.

Expression Vectors

used to express full length cDNA in bacteria. It contains a promoter and other signals for efficient transcription and translation. Thousands of cDNA libraries have been constructed from many different tissues or stages of development from many organisms.

Library Screening

method to ID the clone of interest from a library. Involves mixing probed ss Nucleic acids under proper NaCl and Temp. Probes are radioactively labeled.

Oligonucleotide

are short sequences of nucleotides (RNA or DNA), typically with twenty or fewer bases. Used as PCR Primers.

Southern Blot

Southern blot has limited ability to ID small DNA Mutations; can see gross deletions. Digest DNA with three different restriction enzymes. The fragments are separated according to size within an agarose gel under an electric field. After electrophoresis, the fragments are rendered single stranded and transferred to a membrane by capillary action. The labeled single-stranded probe is applied to the membrane, and the probe is allowed to anneal to its complementary DNA sequences. After unannealed probe is washed off, membrane is placed against an x-ray film. The fragments annealed complementary to the probe are revealed.

Allele Specific Oligonucleotide Probes (ASO)

Can identify small DNA mutations on the order of 1 to a small number of bases. ASO hybridization begins with PCR. Once amplified, the target DNA is hybridized to a membrane in wells (without prior electrophoresis). Oligonucleotide probes specific for normal and mutant sequences are radioactively labeled and hybridized to the substrate-bound target DNA. Competitive binding between mutant and normal probes permits probe binding dependent upon the sequence of the target DNA. Polymorphic sequences near the sequence can cause false positives or false negatives.

Diseases that can be detected with ASO

• Sickle Cell is a mutation in a single base, converting Glutamic Acid to Valine in B-Globin.

• Cystic Fibrosis: 60-70% of all mutations are caused by a 3-base frameshift deletion in the deltaF508 allele of the CFTR; deletion of Phenylalanine.

Best probe to detect a single-base mutation or small insert or deletion is a synthetic oligo as its short length makes it much more sensitive to even single-base pair mismatches.

Northern Blot

Isolate cellular RNA/mRNA from a particular cell type & separate by size on a denaturing formaldehyde agarose gel; the fractionated RNA in the gel is transferred to a membrane and hybridized with a probe; after washing, the membrane is exposed to x-ray film to view the radioactively labeled RNA bands.

PCR: Polymerase Chain Reaction

Alternative to cloning for generating unlimited amounts of a particular sequence. Required: 2 Oligo-Primers, 1 for target strand and another used on the complimentary strand; 3' ends of the primers are directed towards the target seq. Use of a Thermostable DNA Pol. (Taq) is crucial. Allows for synthesis of 2 new strands using the template between the primers and works under high heat conditions, where temperature is used to dentature the ds DNA and then amplify the target sequence. RT-PCR: start with RNA, synthesize cDNA, and use the cDNA as the primer to make more copies.

Quantitative PCR (qPCR)

Measures the amount a PCR product increases during the PCR reaction. The rate of increase is used as a measure of the amount of template present at start. The number of PCR cycles required to reach an arbitrary threshold chosen within the exponential portion of PCR amplification is a measure of how much template was initially present when the PCR reaction was initiated.

DNA Sequence Analysis aka 'Sanger Method' or 'Sanger Sequencing'

Use ddNTs which are radioactively labeled and lack the 3'hydroxyl group (and 2'-OH) which doesn't allow elongation. DNA fragment to be sequenced is used as a template and is primed by a short Oligo and extended by DNA Pol. Followed by the addition of ddNTs with 4 normal NTs. The strands are separated using electrophorsis.

DNA Sequence is read from the 1st NT eluted to the last (typically bottom-up).

Microarray

A wafer which has thousands of wells (referred to as an array "chip").

Nucleic acids are spotted individually in a matrix pattern and hybridized with probes. Measures differential gene expression or DNA copy number. Can use probes ranging from short 25 basepair oligos to BAC clones with 350kb.

FISH (Fluorescence In Situ Hybridization)

Used to visualize chromosomal aberrations such as deletions, duplications, and translocations.

Probes are labeled with fluorescent dyes and hybridized to DNA within chromosomes which are immobilized on microscopic slides from Interphase (chromatin is diffuse) or Metaphase (chromosomes) and denatured in place ("in situ"); gene specific probe.

Chromosome "Painting" Probes

complex mix of DNA from all or part of a chromosome used as a probe to stain some or all of a chromosome

Spectral Karyotyping (SKY)

uses 24 different chromosome painting probes each labeled with a different dye.

Can see abnormal chromosomes consisting of pieces of different chromosomes; view altered structures, including translocations, large/gross deletions. Many chromosomal abnormalities can be viewed in late stage tumor cells. In females, only 23 colors present because of the absence of the 24th Y chromosome.

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