Lecture 16: Analyzing Cell, Molecules and Systems 2

Question 1

The human genome project was finished in 2004 with ___ billion nucleotides - 1 copy or 23 chromosomes

Answer 1

3 billion nucleotides

Question 2

The human genome has how many genes

Answer 2

26,000 ( the smae as in mustard plant or earthworm)

Question 3

__% of the human genome is responsible for coding

Answer 3

1.5%

Question 4

Restriction endonucleases function, examples, and where the examples cut

Answer 4

• These are isolated from bacteria and cut DNA at specific nucleotide sequences
• Examples
  
  • Haelll: cuts at a sequence of four nucleotide pairs
    
    • cuts straight through the two strands
  • EcoRI: makes a staggered cut and generates “sticky ends”
    
    • makes cut in in front of ‘AATT’ sequence
  • Hindlll: makes a staggered cut and generates “sticky ends”
    
    • makes cut in front of AGCTT sequence

Question 5

How is Agaros gel different from SDS-PAGE

Answer 5

• Agarose gel is a form of electrophoresis that is used to analyze DNA
• since DNA has a negative charge it does not require the negative detergent that SDS-PAGE needs (SDS-PAGE is used on proteins)

Question 6

genes can be cloed using bacterial plasmid by

Answer 6

• The plasmid is cut open with a restriction nuclease
• Then DNA fragment is mixed in
• DNA ligase and ATP are added to close the nick.

Question 7

DNA ligase can join together any two DNA fragments in vitro to produce

Answer 7

• recombinant DNA molcules ( this is much easier with compatible cohesive ends)
• (note if joining two fragments cut by different restriction nuceleases then you need DNA + dNTPs to make the ends compatible)

Question 8

DNA libraries

Answer 8

• Every gene in the human body can be put into bacteria and sotred indefinitely and propagated at anytime
  
  • because the DNA fragments were derived directly from the chromosomal DNA of the organism of interest, the resulting colleciton- called a genomic library- wil represent the entire genome of that organism
  • human genomic libraries containing DNA fragments that represent the whole human genome can be constructed using restriction nucleases and DNA ligase

Question 9

cDNA library

Answer 9

• a DNA library that enriches for protein-coding genes
• begins the cloning process by selectign only those DNA sequences that are transcribed into mRNA thus correspond to protein-encoding genes. This is done by extractign the mRNA from cells and then making a DNA copy of ach mRNA molecule present
• the copying reaction is catalyzed by the reverse transcriptase enzyme of retroviruses, which synthesizes a complementary DNA chain on an RNA template
• they are then converted by DNA polymerase into double-stranded cDNA molecules, and inserted into plasmid or virus vector and cloned
• cDNA clones
  
  • no introns
  • much smaller than original gene
  • requires viral enzyme
    
    • Reverse transcriptase

Question 10

Difference in DNA libraries

Question 11

FISH

Answer 11

• Fluorescence in situ hybridization (FISH)
• can be used to analyze the presence and location of genes - cytogenetics
• Molecule cna udnergo denatruation and renaturation (hybridization)
  
  • this is done using heat to denature and then slowingly cooling to renature DNA
• With FISH DNA is Denatured and then DNA probes are used to label the DNA because the denaturation allows them base-pari with their complementary sequences.

Question 12

Polymerase chain reaction (PCR)

Answer 12

• PCR allows DNA from a specified region- selected by the experimenter- to be greatly amplifed, effectively “purifying” this DNA away from the remainder of the genome, which remains unamplified.
  
  • this is done using DNA primers that are designed to uniquely locate any position of a genome
    
    • these primers mark the right and left boundaries of the DNA to be amplified
• DNA primers
  
  • small (about 20 nucleotides)
  • complimentary to some portion of single stranded DNA

Question 13

PCR is used in forensic science to distinguish one individual from another. Explain how

Answer 13

• the DNA sequences analyzed ar short tandem repeats (STRs) composed of sequences such as CACACA….. or GTGTGT
  
  • STRs are found in various positions in the human genome.
  • The number of repeats in the STRs is highly variable ranging from 4-40
  • individuals usually inherit a number a different number of repats at each STRs locus from their mother and father.
• The primers are able to identify the STRs

Question 14

Alternations in gene expression or presence of foreign DNA can be detected by

Answer 14

• PCR (used to detect HIV more specifically qPCR is used)
• PCR can be used ot detect the presence of a viral genome in a sample of blood
• becuase of its ability to amplify enormously the signal form a single molcule of nucleic acid, PCR is an extraordinarily sensitive method for detecting trace amounts of virus in a smaple of blood or tissue

Question 15

What is qPCR

Answer 15

• quantitative PCR
• is able to show the level of change present

Question 16

PCR is used to detect what pathogens

Answer 16

• Chlamydia, cytomegalovirus (CMV), Hep C, Myobacterium tuberculosis, Neisseria gonorrhoeae

Question 17

qPCR is used to detect what pathogens

Answer 17

Streptococcus and HIV

Question 18

Hybridization is used to detect what pathogens

Answer 18

Trichomonas vaginalis and Gardnerella vaginalis

Question 19

Inherited disorders diagnosed via PCR

Answer 19

• Cystic fibrosis’
• Duchenne muscular dystrophy
• Glucose 6- phosphate dehydrogenase deficiency

Question 20

There are 1 in _____ nucleotide differences between two human genomes

Answer 20

1 in 1,000 nucleotide differences

Question 21

Single-nucleotide polymporphism (SNPs)

Answer 21

• A variation between individuals in a population due to a relatively common difference in a specific nucleotide at a defined point in the DNA sequence
• can be neutral, pathogenic, or predisposing

Question 22

if you want to measure the activity of every gene in a cell or tissue you can use a device called a

Answer 22

• DNA microarray chip
  
  • device is a glass square
  • there are small microsquares or spots on this device
  • each spot has a different DNA sequence (25nt) from a different gene
  • 26,000 genes: so spot 1= gene 1
    
    • spot 2= gene 2
• probes: can take RNA from cells or tissue, make DNA using reverse transcriptase and add a fluorescent marker
• inject into the chip and the probe will bind with its complementary sequence
• Scan- scanner will measure intensity fo spot and tell how much gene expressed
  
  • more intense signal= higher degree of binding of probe= higher level of expression
• 20 spots per gene (25 nts from different parts of gene)

Question 23

Example of how microarrays can be used

Answer 23

• Tumor found and surgcally removed
• chemotherapy follows
• tumor returns and chemotherapy not effective
• you can use the microarrya analysis that was made on the tumor to see why