Chapter 21 Manipulating genomes

define genome

all the genetic material an organism contains

define introns

non-coding DNA

98% of DNA

define extrons

coding DNA

2% of DNA

define satellite DNA

repeating sequences of DNA within introns

define minisatellite

20-50 bases in length repeated 50-100s times

occur in more than 1000 locations in the human genome

known as variable number tandem repeats (VNTRs)

define microsatellite

2-4 bases in length repeated 5-15 times

known as short tandem repeats (STRs)

define DNA profiling

producing an image of the patterns in the DNA of an individual

stages of DNA profiling

1) extraction = cheek swab, blood sample = a lot of DNA is needed = polymerase chain reaction (PCR) used to amplify DNA sample

2) digestion = restriction endonucleases cut DNA at specific restriction sites = fragments

3) separation of DNA fragments = electrophoresis

4) hybridisation = radioactive or fluorescent DNA probes added to DNA fragments

5) development = radioactive labels added, x-ray images taken = fluorescent labels added, placed under UV light so fluorescent tags glow

define DNA probes

short DNA/RNA sequences complementary to a known DNA sequence

describe electrophoresis

1) DNA is negative (PO₄^3-)

2) samples of DNA fragments are pipetted into wells and encased in agarose gel

3) DNA moves to the positive terminal through a mesh

4) longer DNA fragments take longer to reach the positive terminal, shorter fragments move faster

5) creates bands of DNA

steps of PCR

1) denaturation of DNA = 95°C

2) annealing of the primers = 55°C =primers bind to ends of DNA strands

3) extension = 72°C = TAQ polymerase produces double stranded DNA identical to the original sequence

uses of genomes

1) identifying presence/vulnerability to disease

2) identifying species

3) identifying evolutionary relationships

define DNA sequencing

the process of determining the precise order of nucleotides within a DNA molecule

describe the development of DNA sequencing

Frederick Sanger developed techniques for sequencing nucleic acid from viruses

Sanger sequencing technique involved radioactive labelling of bases and gel electrophoresis

development of this technique led to capillary sequencing that was used in the HGP

describe the human genome project

1990-2003

scientists mapped entire human genome

steps of DNA sequencing

1) DNA is mixed with a primer, DNA polymerase, and A/T/C/G/terminator bases

2) mixture placed on thermal cycler = PCR steps

3) nucleotide/terminator bases are randomly added to the ssDNA

4) if a terminator base is incorporated into the newly synthesised strand, DNA synthesis is terminated as no more bases can be added

5) DNA fragments are separated based on length by capillary sequencing = fluorescent markers on terminator bases are used to identify final base on each fragment

why is genome sequencing quicker today

massively parallel sequencing uses flow cells which forms clusters of identical DNA fragments which are all sequenced and imaged at the same time

requires highly developed technology

fast and cheap = more genomes can be sequenced

define bioinformatics

the development of software and computing tools needed to organise and analyse raw biological data (algorithms, mathematical models, statistical tests)

define computational biology

using data from bioinformatics to create theoretical models of biological systems which can be used to predict what might happen if conditions changed

define genomics

an interdisciplinary field of science focusing on the structure/function/evolution/mapping/ and editing of genomes

define proteomics

the study of amino acids of an organisms entire protein complement

define synthetic biology

the design and construction of novel artificial biological pathways/organisms/designs or the redesign of existing natural biological systems

what techniques is synthetic biology used for

genetic engineering, gene therapy, industrial context (e.g. production of drugs), synthesis of new organisms

how can 20-25000 coding genes form a larger number of proteins

splicing, protein modification

2 ways a gene can be isolated

restriction endonucleases cut the required gene at specific restriction sites = cutting the two DNA strands unevenly leaves unpaired, exposed bases called sticky ends

isolate the mRNA for the gene from a cell that produces the required polypeptide product = uses reverse transcriptase and DNA polymerase to create DNA

define genetic engineering

the manipulation of the DNA sequences of an organism

define recombinant DNA

altered DNA in the host cell with introduced nucleotides

define transgenic organism

contains nucleotide sequences from a different species