Genetic Engineering Technology Midterm 3 Lecture 1

Front 1

Genome

Back 1

the entire genetic content within an organism

Front 2

Genomics

Back 2

involves the determination of the content, organization, and evolution of the genomes of different organisms
o Genomics is closely related to bioinformatics, which uses computers, computational tools, and databases to organize and analyze DNA and protein information

Front 3

The goals of Genomics:

Back 3

• 1. Assembly of maps of the genomes of different organisms
• 2. Compilation and organization of both expressed gene sequences and other non-expressed regions
• 3. The generation of gene expression profiles under different conditions
• 4. Finding the location of all genes in a genome and annotating each gene
• 5. Determination of gene function and regulation (functional genomics)
• 6. The identification of all proteins in a genome and their functions (proteomics)
• 7. Characterization of DNA polymorphisms within the genome of a species
• 8. The comparison of genes and proteins in the genome of one species with genomes of other species (Comparative Genomics)
• 9. The implementation and management of databases and genome-based research tools accessed through the Internet
o These goals were lofty when set, yet most are well underway and a few have been completed
o Many goals depend on having a map of the organism’s genome

Front 4

Genetic linkage v. Physical maps

Back 4

•Genetic linkage maps determine the relative arrangement and approximate distance between genes and markers on the chromosome
 Analogous to a road map that shows city locations and other landmarks (very vague, low resolution)

•Physical maps give the physical location on the chromosome in base pairs, and the distance between genes or DNA fragments of unknown function
 Analogous to a street map that shows the location of houses on a street (very specific, high resolution)

•Cytological maps bridge the gap between the two (medium resolution)

Front 5

Genetic Linkage Maps

Back 5

o Show the order and distance between pairs of linked genes, which are genes on the same chromosome
o Arrange genes on a chromosome based on how often two genes are involved in homologous recombination
o During meiosis, each human chromosome is involved in 1.5 crossover events
o Likelihood of crossing over increases as distance between two genetic loci increases
o Crossover events rare between linked genes
o Distances measured in centiMorgans (cM)
o 1 cM = 1% crossover rate = ~106bp or 1 Mb

Front 6

contigs

Back 6

sets of ordered, overlapping cloned DNA fragments

contiguous stretches of DNA

Front 7

Cytological maps

Back 7

o Help to align genetic linkage maps with the more specific physical map
o Involved the banding pattern of a chromosome through karyotyping–the staining and viewing of a chromosome
o Used to diagnose human diseases caused by chromosomal abnormalities such as Down syndrome (3 copies of Ch21)
o Can also use FISH (fluorescence in situ hybridization) to locate specific genes or markers on chromosomes
o Fluorescent-tagged DNA probe added to a slide with metaphase chromosomes on it and viewed with epifluorescence microscopy

Front 8

karyotyping

Back 8

–the staining and viewing of a chromosome

Front 9

FISH

Back 9

fluorescence in situ hybridization

Front 10

Physical Maps

Back 10

o Assembled from contigs (contiguous stretches of DNA)
o Distances between genes and landmark DNA sequences are shown in kilobases (kb)
o Provide information about the physical organization of a piece of DNA
• Example: Location and types of restriction enzyme sites within a stretch of sequence
o Ultimate physical map is the complete DNA sequence of the human genome, which is about 3 billion bp
o Because many genomes are huge, DNA must be cut into fragments and cloned into YACs, BACs, etc. to make the contigs to be aligned
o A universal reference system has been developed to help align contigs using sequence-tagged sites (STSs)
o STSs are simple short stretched of sequenced DNA

Front 11

Resolution of maps

Back 11

o The lowest resolution map is the genetic linkage map
o The highest is the physical map

Front 12

Why was the physical mapping of the human genome was quite a daunting task:?

Back 12

• Human genome has 30,000 –40,000 genes
• 23 pairs of chromosomes (22 autosomal & 1 sex pair)
• 3 billion base pairs of sequence
• Smallest chromosome (Y) is 50 million bp
• Largest chromosome (1) is 250 million bp
• ONE human cell has 5 feet of DNA!

Front 13

Funding of the HGP

Back 13

o National Institutes of Health (NIH) and Department of Energy (DOE) developed an action plan for the HGP in 1988, which began in 1990
o NIH/DOE spearheaded a public group to complete the HGP
o US Congress provided funds to the Nat’l Center for Human Genome Research (NCHGR) and DOE:
o 50% to studying the human genome
o 25% to studying the genomes of other organisms
o 20% to conferences, training, and program admin’n
o 5% to the consideration of ethical issues

Front 14

Goals of the HGP

Back 14

o Originally divided into 2 five-year plans, but altered as it progressed:
• 1. Generation of high-resolution genetic and physical maps that will help locate genes implicated in disease
• 2. Establishment of DNA sequence landmarks that will help complete the entire genome sequence by 2005
• 3. Identification of all the genes in the genome using bioinformatics, ESTs, and comparative genomics
• 4. Establishment of polymorphism databases that will aid in disease diagnostics, variations in global human populations, and understanding human evolution

Front 15

HGP required new technology

Back 15

o Such lofty goals required all sorts of new technologies and methods
• DNA Sequencing (dideoxy tags)
• Also studying gene expression, bioinformatics, and establishment of informational databases

Front 16

HGP comes of age

Back 16

o 1994 –First high-resolution physical map for the human genome was published
o 2001 –The public human genome sequence draft was published
o 2003 –Close to 99.9% of the gene-containing portion of the human genome was sequenced to 99.9% accuracy
o “Completion” was 2 full years ahead of schedule
• US Government very pleased because then they could use the funds for something else
• What did they overlook?
 Project not really completed! What does it all mean?

Front 17

Two groups published first drafts in 2001 by different methods

Back 17

o Private group lead by the Celera Corporation used a “shotgun” sequencing approach
• Clones randomly sequenced
• DNA sequence assembled using computer software
o Public group headed by the NCHGR used a clone-by-clone sequencing approach
• Discussed last time (contigs)
o DNA came from anonymous ethnically diverse donors
o Most samples came form males, although females were later used to refine the sequence...Why use male DNA?
• Males have both X and Y

Front 18

Both HGP groups had similar findings:

Back 18

• Human genome contains a little more than 3 billion base pairs (3164.7 million nucleotides)
• Found 1/3 number of genes expected (~30,000 –40,000, not 80,000 –100,000)
• Only approximately 1.5% of the human genome encodes for the production of proteins
• Repeated sequences that don’t encode protein make up at least 50% of the genome
• The average length of gene is ~3000 nucleotides, but size varies greatly
• Genes are not evenly distributed, but concentrated in certain areas
• Approximately 200 genes originated directly from bacteria
• Human proteins are more complex than proteins with similar functions in lower organisms
• The germline mutation rate in males in twice as high as in females
• The nucleotide sequence in all humans in 99.9% identical
• More than 50% of newly discovered genes have unknown functions
• Very interesting things appear when genomes compared