Term
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Definition
an accurate sequence of the human genome was completed in 2003
by 2013 the genomes of >7000 species have been sequenced |
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Term
| THE GENERAL IDEAS BEHIND GENOME SEQUENCING |
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Definition
| fragment the genome, cloning DNA fragments, sequencing DNA fragments, reconstructing the genome sequences from fragments, and analyzing the infor found in genome |
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Definition
| recognizes sequences of bases anywhere within the genomes. cuts sugar-phosphate restriction fragments are generated by digestion of DNA with restrict enzymes |
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| RECOGNITION SITES FOR RESTRICTION ENZYMES |
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Definition
are usually 4-8 bp of double-stranded DNA
often palindromic- read 5'-3'
cuts at same place relative to its specific recognition |
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| cuts are straight through both DNA strands at the line of symmetry |
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| cuts are displaced equally on either side of the line of symmetry |
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| is 4^n where n is the number of bases in the recognition site |
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| average restriction fragment is 256 bp |
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| average restriction fragment size is 4100 bp |
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Definition
can be used to fragment DNA at random locations
can break phosphodiester bonds
pass DNA through a thin needle and sonication |
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Definition
distinguishes DNA fragments according to size
place in buffered solution and send electrodes through gel so charged particles move negative to positive |
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| AFTER GEL ELECTROPHORESIS |
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Definition
| visualize DNA fragments by staining gel with fluorescent dye, and photograph gel under uv light |
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Definition
| genomes of animals, plants and microorganisms are too large to analyze all at once |
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Definition
| is a means to purify a specific DNA fragment away from all other fragments and make many identical copies of that fragment |
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Definition
insert DNA fragments into cloning vectors to make a recombinant DNA molecule
transport recombinant DNA into living cells to be copied |
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Term
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Definition
| cuts are straight through both DNA strands at the line of symmetry |
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Term
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Definition
| cuts are displaced equally on either side of the line of symmetry |
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Term
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Definition
| is 4^n where n is the number of bases in the recognition site |
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Term
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Definition
| average restriction fragment is 256 bp |
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Term
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Definition
| average restriction fragment size is 4100 bp |
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Term
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Definition
can be used to fragment DNA at random locations
can break phosphodiester bonds
pass DNA through a thin needle and sonication |
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Term
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Definition
distinguishes DNA fragments according to size
place in buffered solution and send electrodes through gel so charged particles move negative to positive |
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Term
| AFTER GEL ELECTROPHORESIS |
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Definition
| visualize DNA fragments by staining gel with fluorescent dye, and photograph gel under uv light |
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Term
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Definition
| genomes of animals, plants and microorganisms are too large to analyze all at once |
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Term
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Definition
| is a means to purify a specific DNA fragment away from all other fragments and make many identical copies of that fragment |
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Term
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Definition
insert DNA fragments into cloning vectors to make a recombinant DNA molecule
transport recombinant DNA into living cells to be copied |
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Term
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Definition
cloning vectors have 2 main features:
- origin of replication
- restriction sites(s) for clonin insert DNA
- selectable marker ex-> antibiotic resistance |
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Term
| ALTERNATE VECTORS THAT CAN CARRY LARGE INSERTS |
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Definition
| bacterial artificial chromosomes and yeast artificial chromosomes |
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Term
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Definition
| of the vectors and human genomic DNA with a restriction enzyme results in complementary sticky ends |
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Definition
| is used to seal the phosphodiester backbones between vector and inserts |
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Definition
| host cells take up and amplify recombinant DNA |
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Definition
| the process by which a cell organism takes up foreign DNA |
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Definition
| only .1% of cells will be transformed with plasmid |
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Definition
| only the cells with plasmid will grow on on media with ampicillin |
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Definition
| multiplies to produce millions of genetically identical cells, each with recombinant plasmid |
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Definition
| long-lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector |
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Term
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Definition
| contains a different recombinant plasmid, each with a part of the human genome |
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Term
| A PERFECT GENOMIC LIBRARY |
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Definition
has one copy of every sequence in the entire genome.
# of clones= length of genome/avg size of inserts |
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Term
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Definition
| number of clones in a perfect library |
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| IMPOSSIBLE TO OBTAIN A PERFECT LIBRARY |
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Definition
-usually libraries are made that have 4-5 genomic eqivalents
- gives an avg of 4-5 clones for each locus |
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Definition
| uses DNA polymerase to make new DNA |
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Definition
| a single strand of DNA to copy |
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Definition
-short single stranded DNA molecule that is complementary to the template
- designed to be complementary to plasmid sequence adjacent to the unknown insert |
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Definition
-is a good template for sanger sequencing
- template and primer interact through hybridization |
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| SANGER SEQUENCING GENERATES |
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Definition
-a series of single stranded DNA fragments
- fragments include primer and nucleotides complementary to unknown DNA |
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Term
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Definition
| they each differ in length from proceeding and succeeding fragment by one nucleotide |
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Term
| DIDEOXYNUCLEOTIDE (ddNTP) |
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Definition
-terminates DNA synthesis
-labeled with a different fluorescent dye for detection of the sequence |
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Term
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Definition
| separated by size using electrophoresis |
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Term
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Definition
| migrate quickly and appear at the bottom of the gel |
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Term
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Definition
| after electrophoresis, fragments flow through a detector and the color of the fragment is digitally recorded |
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Term
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Definition
reads of sequence complementary to the template strand
read 5'-3'
ambiguity recorded as "N" |
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Term
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Definition
began using the hierarchical strategy
BAC genomic library
identify overlapping BAC clones
Shear DNA from each BAC
sequence DNA -assemble sequences 4 overlap |
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Definition
| developed the whole-genome shotgun strategy for genome sequencing |
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Definition
to make shotgun (uncharacterized) clones
shotgun approach can be highly automated |
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Definition
-was partially sequenced from both ends
- pairs of sequence gives spatial information
- the ends were sequenced from 3 libraries with different insert sizes |
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Definition
| helps overcome problems due to repetitive DNA |
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Definition
-may be part of a protein-coding exon
-DNA can be read in 6 reading frames
-ORF is uninterrupted by stop codons |
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Term
| SPECIES RELATEDNESS AND GENOME CONSERVATION |
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Definition
-branch points represent a series of nested common ancestors
-# at each branch point is million years before the present |
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| DNA SEQUENCES THAT ARE CONSERVED |
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Definition
in highly divergent species may be part of a gene
human vs zebrafish -> genome 2% conserved and protein coding is 82% conserved |
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Definition
| can be discovered using a computerized visualization tool |
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Definition
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Definition
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| LOCATING TRANSCRIBED REGIONS |
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Definition
| is a direct method of finding genes |
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| mRNA's ARE NOT EASILY SEQUENCED |
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Definition
to rare to purify
sequencing technology not widely available |
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Term
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Definition
cDNA for sequencing
retroviruses have RNA genomes
they use reverse transcriptase to copy RNA into cDNA |
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Term
| CONVERTING RNA TRANSCRIPTS TO cDNA |
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Definition
obtaining mRNA from red blood cell precursors
eukaryotic mRNA poly A tails at 3' end |
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Term
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Definition
| affinity to oligo (dT)- single strand DNA fragments of 20 nucleotides made of dT only |
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| CONVERTING RNA TRANSCRIPTS TO cDNA |
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Definition
synthesis of hybrid cDNA-mRNA molecule
in vitro synthesis using reverse transcriptase + dATP+ dGTP+dTTP+cCTP |
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Term
| CREATING THE 2ND DNA STRAND COMPLEMENTARY TO THE FIRST cDNA STRAND |
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Definition
mRNA digested with RNAse
3' end of cDNA folds back and acts as primer for 2nd strand |
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Term
| PRESENCE OF dNTP's AND DNA POLYMERASE |
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Definition
| the first cDNA strand acts as a template for synthesis of the second cDNA strand |
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Term
| THE ARRANGEMENT OF GENES ON GENOME |
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Definition
human genome has about 25000 genes
part of genome that corresponds to exons is exome |
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| MOST OF A GENOME IS NON-CODING DNA |
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Definition
exome= 2%
introns
centromeres, telomeres, transposable elements
simple repeating sequences |
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Term
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Definition
-be transcribed in the same or opposite directions
- both chromosomal strands are the template strand for some genes |
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Term
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Definition
| chromosomal regions that have many more genes thatn expected form avg gene density over entire genome |
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Term
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Definition
regions of >1 Mb that have no identifiable gene
3% human genome is comprised of gene desert |
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| CLASS III REGION OF THE HUMAN MAJOR HISTOCOMPATIBILITY |
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Definition
MHC complex contains 60 genes within a 700kb region
GC content is much higher than genome avg |
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Term
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Definition
| protein domains- sequence of amino acids that fold into functional units |
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Definition
| protein products have novel domain architechtures |
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Definition
| are groups genes closely related in sequence and function |
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Definition
| look like genes, but do not function as genes |
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Definition
| arose from the same gene in the common ancestor, usually retain same function |
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Definition
| arise by duplication, often refers to members of a gene family |
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Definition
| colored segments contain at least 2 genes whose order is conserved in the mouse genome |
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| CHROMOSOMAL REARRANGEMENTS |
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Definition
| have moved the blocks to different places in the mouse genome |
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Term
| HUMAN T CELL RECEPTOR FAMILY |
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Definition
DNA rearrangement combines V,D, and J segments into a gene
result is about 1000 different combinations |
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Term
| COMNINATIORIAL STRATEGIES AT THE RNA LEVEL |
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Definition
may lead to gene amplification and diversity
example-> 2 neurexin genes
2 alternative promoters, 5 sites for alternative splicing
can generate 2000 different mRNA's |
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Term
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Definition
database established by the NIH in 1982
still the most widely used repository for sequence data |
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Term
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Definition
single, complete, annotated version of a species'genome
agreed upon standard form comparison
maintained by the NCBI |
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Definition
| find homologous sequences |
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Term
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Definition
carries oxygen in the blood
adult hemoglobin
2 alpha and 2 beta |
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| EMBRYONIC AND FETAL HEMOGLOBINS |
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Definition
| bind more tightly to oxygen to facilitate transfer of oxygen from the mother to the embryo or fetus |
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Definition
| binds to oxygen less tightly to allow delivery of oxygen to the organs |
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Term
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Definition
genes in order on chromosome
genes oriented in same direction
locus controls region-> controls sequential expression of globin genes |
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Term
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Definition
deletion of alpha and beta genes
y genes continue to be expressed
normal levels of health |
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Term
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Definition
change in amino acid sequence of a- or b-globin chain
causes destruction of red blood cells
example sickle cell |
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Term
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Definition
mutations reduce or eliminate production of one of the two globin polypeptides
range of phenotypes |
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