biotech

Question 1

DNA

Answer 1

deoxyribonucleic acid

Question 2

polymerase chain reaction

Answer 2

enables small quantities of DNA to be replicated to make vast amounts of DNA (testable amounts to be use in analysis techniques (DNA amplification)

Question 3

steps of PCR

Answer 3

denaturing: two strand of DNA separate
annealing: short sections of DNA are extended to produce longer strands
extension /synthesis: short sections of DNA are extended to produce longer strands

Question 4

denaturing

Answer 4

original DNA is heated to 95°C for 5 mins to break down hydrogen bonds holding two strands together so it separates into two single strands of DNA

Question 5

annealing

Answer 5

solution is cooled to 55°C and allows primer to bind to complementary bases sequence on each of single strands of DNA, provides starting point for DNA replication

Question 6

elongation/extension

Answer 6

solution is heated to 72°C and heat resistant DNA polymerase enzyme taq catalysed synthesis of of complementary strand for each of sin glue strands of DNA using supply of free nucleotides. DNA polymerase produces two identical double strands of DNA

Question 7

Components of PCR

Answer 7

original sample of DNA is dissolved in solution and mixed with

• DNA polymerase: heat stable form of enzyme extracted from thermophilic bacterium: taq polymerase
• 4 nucleotides: guanine, cytosine, adenine, thymine
• DNA sample
• primers
• mix buffer

Question 8

gel electrophoresis

Answer 8

techniques used to separate fragments of DNA according to length (use gel and electric current)

DNA sample is extracted from tissue of dead or living organism and treated with particular restriction enzyme: cuts strands of DNA at a specific sequence of nucleotides

DNA fragments in solution are made visible by staining with methylene blue or fluorescent dye and placed in wells at end of agar plate

Question 9

summary of steps of gel electrophoresis

Answer 9

1. Restriction enzymes break DNA into smaller segments of various sizes
2. DNA segments are loaded into wells of gel, gel floats in buffer solution
3. Electric current is passed through chamber, DNA fragments move toward positive electrode (away from negative as DNA has negative charge)
4. smaller DNA segments move faster and farther than large DNA

Question 10

genetic fingerprinting

Answer 10

DNA fragments used are short tandem repeats (sections of non coding DNA with many repeating bases that differ between individuals

Question 11

filling wells

Answer 11

depressions (indents) in gel so when current is applied DNA will move through gel rather than diffuse through solution. DNA must be placed accurately in wells done using micropipette

Question 12

visualising DNA

Answer 12

methylene blue dye binds to DNA when gel is soaked in dye areas containing DNA stains darker blue and is visible to naked eye

DNA probe: short sections of DNA with radioactive or fluorescent molecules that binds to DNA being tested exposes film and produces visible pattern of light and dark bands on a piece of film

Question 13

restriction enzymes

Answer 13

occur naturally in bacteria protect the bacterial cells from infection by foreign DNA by cutting DNA into smaller pieces. Restriction of infection by viral DNA

Question 14

characteristics of restriction enzymes

Answer 14

cuts DNA at specific base sequence recognition sites (4-8 base pairs in length
- palindrome: base sequence recognition is same thing backward

Question 15

ends of restriction enzymes

Answer 15

• blunt ends: when recognition sites cut DNA between two specific bases at recognition
• sticky ends: when restriction enzymes cut DNA in a staggered manner at a recognition site results in overhangs on two cut ends of DNA, able to bind to complementary sticky ends on other DNA

Question 16

recombinant DNA

Answer 16

involves transferring DNA from one organism to another, DNA that has had a gene from one organism is isolated and transferred to another organism, segment of extracted DNA combines with DNA of second organism producing a transgenic organism

Question 17

transgenic organism

Answer 17

an organism that had DNA from another species introduced into it artificially. Produced for production of specific proteins e.g insulin, vaccines, antiviral medication

Question 18

production of recombinant DNA

Answer 18

• DNA enzymes
• restriction enzymes
• ligases: catalyse the recombining or joining of DNA fragments (ligation)
• exposed nucleotides: are attracted by base pairings to another fragment
• DNA ligase: joins fragments at their sugar phosphate backbones
• plasmids: bacteria contain large strand of chromosomal DNA which holds main genetic info also has several smaller rings of double stranded DNA (plasmids), contain genes that produce protection against antibodies from other bacteria, use as vectors to transfer genetic info

Question 19

steps in reproducing recombinant DNA

Answer 19

• required gene located and cut from DNA molecule using specific restriction enzyme at specific recognition sites that can produce blunt or sticky ends with jagged cut which create overhang
• bacterial plasmid which is the vector is isolated and removed from bacterium using same restriction enzyme as used in step 1, which ensures ends are complementary to ends of required gene
• required gene is joined to plasmid using DNA ligase which joins sticky ends together
• recombinant DNA is returned to/ inserted into bacterial host cell
• bacteria rapidly synthesises and replicates vast quantities forming a transgenic organism

Question 20

DNA sequencing

Answer 20

determining precise order of nucleotides bases in DNA, used to sequence length of DNA and identify start of gene where primers can attach

Question 21

Sanger sequencing method

Answer 21

1. DNA sample is amplified: heated to 95° to denature and separate DNA strands and produce template and complementary strand for sequencing
2. primer is annealed to the 5’ end of DNA
3. primed DNA is divided equally among 4 test tubes and taq polymerase is added to all 4 tubes as well as all 4 dntps and modified ddntps (only one of each type is added to test tube)
4. Taq attaches dntps to template strand starting at primer until ddntp is is added, once this occurs sequence is terminated due to lack of OH in ddntp: causes DNA fragments to be different lengths
5. Gel electrophoresis is used to sequence DNA, poured into 4 different wells and electric current is passed through gel, (shorter = faster)
6. Sequence is read from bottom of plate to top and results in complementary sequence of sample DNA

Question 22

human genome project

Answer 22

purpose: identify all genes in human DNA and determine sequence of 3 million base pairs that makeup human DNA
- resulted in:identifying location of 4000 potentially faulty genes, identifies genes that cause disorders, enabled monitoring of genes involved in diseases
- development of genetics test for screening diseases and allowing people to be given steps in order to modify their lifestyle to prevent disease occurring

Question 23

gene therapy

Answer 23

treatment of genetic disorders by gene therapy targets replacement of defective genes that causes diseases can potentially replace faulty genes, provide different genes that will perform correct function

Question 24

steps in gene therapy

Answer 24

1. Body cells from patient are isolated
2. Copy of normal human allele is inserted into DNA vector
3. Body cells are infected with virus containing recombinant DNA
4. Viral DNA carrying normal allele inserts itself into patients chromosome
5. Cultured cells are injected into patient, presence of normal cells / alleles relieves patients symptoms